Risk Evaluation for 1,4-Dioxane Systematic Review Supplemental File: Data Quality Evaluation of Human Health Hazard Studies, Animal and In Vitro Studies CASRN: 123-91-1 December 2020


United States
hi	Environmental Protection Agency

Office of Chemical Safety and
Pollution Prevention

Risk Evaluation for
1,4-Dioxane

Systematic Review Supplemental File:

Data Quality Evaluation of Human Health Hazard Studies,
Animal and In Vitro Studies

CASRN: 123-91-1

December 2020

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EPA's Office of Pollution Prevention and Toxics (OPPT) developed data quality criteria for
animal and in vitro studies, presented in the Application of Systematic Review in TSCA Risk
Evaluations document (EPA Document #740-Pl-8001). This document presents data quality
evaluation results for animal and in vitro studies evaluated for the 1,4-Dioxane Risk Evaluation.

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Table Listing

Acute (<24 hr)

1	Animal toxicity evaluation results of Drew et al 1978 for a 4-hour inhalation study

on clinical chemistry/bio chemical outcomes (hepatic enzymes) 	 4

2	Animal toxicity evaluation results of Uno et al 1994 for an acute oral study on
mechanistic (gene expression/omics, genotoxicity) outcomes	 8

3	Animal toxicity evaluation results of Mattie et al 2012 for a 6-hour inhalation
study on neurological/behavioral outcomes	 11

4	Animal toxicity evaluation results of Mattie et al 2012 for a 6-hour inhalation sys-
temic effects study on hepatic, renal, irritation, respiratory, body weight outcomes 14

5	Animal toxicity evaluation results of Dow et al 1989 for a single dose in vivo DNA
synthesis study on hepatic, genotoxicity, body weight outcomes	 17

Short-term (1-30 days)

6	Animal toxicity evaluation results of Goldberg et al 1964 for a 10-day inhalation
study on neurological/behavior, body weight outcomes	 20

7	Animal toxicity evaluation results of Roy et al 2005 for an in vivo micronucleus
assay - main study on genotoxicity outcomes	 24

8	Animal toxicity evaluation results of Roy et al 2005 for an in vivo micronucleus
assay - range-finding study on genotoxicity, mortality outcomes	 26

9	Animal toxicity evaluation results of Mattie et al 2012 for a 2-week inhalation
study on neurological/behavioral, body weight outcomes	 29

10	Animal toxicity evaluation results of Mattie et al 2012 for a 2-week inhalation
systemic effects study on hepatic, renal, irritation, respiratory, hematological and
clinical chemistry outcomes	 32

11	Animal toxicity evaluation results of Dow et al 1989 for a repeat dose in vivo
DNA synthesis study on hepatic, genotoxicity, body weight outcomes	 35

12	Animal toxicity evaluation results of Itoh 2019 - in vivo genotoxicity assay - mi-
cronucleus test	 38

Subchronic (30-90 days)

13	Animal toxicity evaluation results of Kasai et al 2008 for a 13-week inhalation
study on hepatic, renal, hematology, clinical chemistry, respiratory, body weight,
mortality outcomes	 41

14	Animal toxicity evaluation results of Kano et al 2008 for a 13-week oral toxicity

of 1,4-d in rats and mice study	 45

Chronic (>90 days)

15	Animal toxicity evaluation results of Argus et al 1965 for a cancer bioassay-liver,
kidney, blood study on cancer outcomes	 48

16	Animal toxicity evaluation results of Kociba et al 1974 for a 2-year drinking water
study study on cancer, hepatic, renal, hematological and immune, body weight,
mortality outcomes	 50

17	Animal toxicity evaluation results of NCI et al 1978 for a cancer bioassay- male
rats study on cancer outcomes	 53

18	Animal toxicity evaluation results of NCI et al 1978 for a cancer bioassay- female
rats and male and female mice study on cancer outcomes 	 57

19	Animal toxicity evaluation results of Torkelson et al 1974 for a chronic toxic-
ity/carcinogenicity assay in rats study on mortality, body weight, hematological
and immune, clinical chemistry/biochemical, cancer outcomes	 62

Page 1 of 187

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20	Animal toxicity evaluation results of Kasai et al 2009 for a 2-year cancer bioas-
say study on cancer, mortality, hepatic, renal, respiratory, hematological and
immune, clinical chemistry/biochemical, nutrition and metabolic/adult exposure
body weight, reproductive outcomes	 65

21	Animal toxicity evaluation results of Argus et al 1973 for a carcinogenicity-liver
(dose response), electron microscopy study on cancer outcomes	 68

22	Animal toxicity evaluation results of Jbrc et al 1998 for a cancer bioassay and
non-neoplastic lesions study on cancer, renal, hepatic, respiratory outcomes .... 70

23	Animal toxicity evaluation results of Kano et al 2009 for a 2-year cancer bioassay
study on cancer outcomes	 72

Developmental

24	Animal toxicity evaluation results of Giavini et al 1985 for a developmental-fetal
effects study on growth (early life) and development outcomes	 74

Genetic toxicity studies

25	Animal toxicity evaluation results of Goldsworthy et al 1991 for nasal epithelium
DNA repair in rats	 76

26	Animal toxicity evaluation results of Kitchin and Brown 1990 for acute rats study

on liver DNA damage 	 78

27	Animal toxicity evaluation results of Yoon et al 1985 for sex linked recessive lethal
mutations in Drosophila study	 81

28	Animal toxicity evaluation results of Kurl et al 1981 for RNA synthesis in rat
liver study	 84

29	Animal toxicity evaluation results for Mcfee et al 1994 for mice bone marrow
micronucleus assay 	 86

30	Animal toxicity evaluation results of Mirkova 1994 for mice bone marrow mi-
cronucleus assay	 89

31	Animal toxicity evaluation results for Miyagawa et al 1999 for DNA synthesis in

rat liver study 	 92

32	Animal toxicity evaluation results of Morita and Hayashi 1998 for mouse liver
micronucleus assay 	 94

33	Animal toxicity evaluation results for Tinwell and Ashby 1994 for bone marrow
micronucleus assay in mice	 97

34	Animal toxicity evaluation results of Morita 1994 for mouse peripheral blood
micronucleus assay 	100

35	Animal toxicity evaluation results of Stott et al 1981 for in vivo DNA synthesis,
alkylation and repair in rats 	103

36	Animal toxicity evaluation results of Fujioka et al 2019 for in vivo mutations in
rats	106

37	In vitro evaluation results of Sina 1983 for mutagenesis in rat hepatocyte assay . 109

38	In vitro evaluation results of Galloway et al 1987 for chromosomal aberration
study in Chinese hamster ovary cells	112

39	In vitro evaluation results of Galloway et al 1987 for sister chromatid exchanges
study in Chinese hamster ovary cells	115

40	In vitro evaluation results of Haworth et al 1983 forbacterial reverse mutation
study	118

41	In vitro evaluation results of Goldsworthy et al 1991 for carcinogenicity in rat
nasal epithelial cells and hepatocytes study	121

42	In vitro evaluation results for Woo et al 1977 for DNA binding assay study .... 124

43	In vitro evaluation results of Nestmann et al 1984 for Ames test study	127

Page 2 of 187

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44	In vitro evaluation results of Zimmermann et al 1985 (194343) for an aneuploidy
study in Saccharomyces cerevisiae	130

45	Animal toxicity evaluation results of Goldsworthy et al 1991 for gavage study in
rats on hepatocyte cell proliferation 	132

46	In vitro evaluation results of Mcgregor et al 1991 for mice lymph cell mutation
assay study	134

47	In vitro evaluation results of Hellmer and Bolcsfoldi 1992 for DNA repair in E.

coli study 	137

48	In vitro evaluation results of Khudoley et al 1987 for bacterial reverse mutation
study	140

49	In vitro evaluation results of Khudoley et al 1987 for bacterial reverse mutation
study	143

50		146

51	In vitro evaluation results of Morita and Hayashi 1998 for sister chromatid exchangel49

52	In vitro evaluation results of Morita and Hayashi 1998 for in vitro micronucleus
study	152

53	In vitro evaluation results of Morita and Hayashi 1998 for mouse liver micronu-
cleus assay mouse lymphoma tk assay (MLA)	155

54	In vitro evaluation results of Morita and Hayashi 1998 for chromosomal aberration
study	157

55	In vitro evaluation results of Morita and Hayashi 1998 for bacterial reverse mu-
tation study	160

56	Animal toxicity evaluation results of Goldsworthy et al 1991 for gavage study in
rats on hepatocyte DNA repair	162

57	In vitro evaluation results of Munoz et al 2002 (195066) for a meiotic non-
disjunction in Drosophila study	164

58	In vitro evaluation results of Sheu et al 1988 for mammalian cell transformation . 166

59	In vitro evaluation report of Stott et al 1981 for bacterial reverse mutation study 169

60	In vitro evaluation results of Dow et al 1989 (4158028) for an unscheduled DNA
synthesis-liver (p 248) study	172

61	In vitro evaluation results of Dow et al 1989 (4158030) for a genotoxicity study

in salmonella	174

62	Animal toxicity evaluation results of Goldsworthy et al 1991 for drinking water
study in rats on hepatocyte DNA repair 	176

63	Animal toxicity evaluation results of Goldsworthy et al 1991 for drinking water
study in rats on hepatocyte cell proliferation	179

64	Animal toxicity evaluation results of Goldsworthy et al 1991 for nasal cell prolif-
eration in rats 	182

Mechanistic

65	In vitro evaluation results of Shah et al 2015 (3115011) for a hepatic CYP450
enzyme activity (metabolism) study	184

66	In vitro evaluation results of Patil et al 2015 for a CYP2el activity in liver mi-
crosomes study	186

Page 3 of 187

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1 Acute (<24 hr)

Table 1: Animal toxicity evaluation results of Drew et al 1978 for a 4-hour inhalation study on clinical chemistry/biochemical
outcomes (hepatic enzymes)

Study Citation: Drew, RT; Patel, JM; Lin, FN (1978). Changes in serum enzymes in rats after inhalation of organic solvents singly and in combination

Toxicology and Applied Pharmacology, 45(3), 809-819
Data Type:	4-hour inhalation

HERO ID:	67913

Domain

Metric

Rating^ MWF* Score

Comments^

Domain 1: Test Substance

Metric 1: Test Substance Identity

Metric 2: Test Substance Source

Metric 3: Test Substance Purity

High	X 2	2 The test substance was identified definitively (by

name).

Low	X 1	3 Test substance source was not reported and a

batch/lot number was not provided; however, the
report states that substances were purchased from
conventional sources and were assayed for purity by
gas chromatography.

High	X 1	1 Test substance purity was reported as >99%.

Domain 2: Test Design

Metric 4: Negative and Vehicle Controls

Metric 5: Positive Controls
Metric 6: Randomized Allocation

Low

Not Rated
Low

x 2

NA
x 1

NA

A concurrent negative control group was tested,
but was not described in detail (e.g., number per
group, treatment method) to allow a determination
of whether it was appropriate and comparable to the
treated groups.

A concurrent positive control group is not necessary
for this study type.

The study did not report how animals were allocated
to study groups.

Domain 3: Exposure Characterization

Metric 7: Preparation and Storage of Test Substance

Medium

x 1

Metric 8: Consistency of Exposure Administration	Medium x 1

The study did not completely report the method and
equipment used to generate the test substance atmo-
sphere; however, there was no reason to believe that
there was an impact on animal exposure. Informa-
tion on storage was not reported; however, there was
no reason to suggest that the test substance was un-
stable.

Details of exposure were reported for the most part
and there was no indication to suggest that the ex-
posures differed among the groups.

Continued on next page

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. continued from previous page

Study Citation: Drew, RT; Patel, JM; Lin, FN (1978). Changes in serum enzymes in rats after inhalation of organic solvents singly and in combination

Toxicology and Applied Pharmacology, 45(3), 809-819
Data Type: 4-hour inhalation

HERO ID: 67913

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 9:

Reporting of Doses/Concentrations

Low

Concentrations were reported as nominal values.
Vapor test concentrations were monitored continu-
ously by an automatic gas sampling gas chromato-
graph; however, actual concentrations were not re-
ported. Due to the lack of reporting of actual con-
centrations for vapor exposures, I downgraded this
metric to low.

Metric 10:

Exposure Frequency and Duration

High

Exposure duration and frequency were reported (4
hours, one exposure) and suitable for the study type
and outcomes of interest.

Metric 11:

Number of Exposure Groups and Dose Spac-

High

The number of exposure groups and concentration

ing

spacing (1000 and 2000) ppm were relevant for the

assessment.

Metric 12:

Exposure Route and Method

Low

The route of exposure (inhalation) was reported and
was suited to the test substance. The method of
exposure was not specifically stated. Additionally,
the number of air changes per hour was not reported,
so I downgraded the score to low.

Domain 4: Test Organism

Metric 13:

Test Animal Characteristics

Medium

The test animal species, strain, sex and starting
body weight were reported; however, age and health
status at the start of the study were not reported.

Metric 14:

Adequacy and Consistency of Animal Hus-

Low

Husbandry conditions (temperature, humidity, light

bandry Conditions

cycle) were not sufficiently reported to evaluate if

husbandry was adequate and similar among the
groups, so I downgraded the score for this metric
to low.

Metric 15:

Number per Group

Medium

The exact number of animals per group was not re-
ported. The authors stated that each experiment
started with 15 animals, , The authors stated that
consecutive daily heart punctures, which were per-
formed to collect blood for serum enzyme assay anal-
yses, resulted in several deaths, but the exact num-
ber of deaths, or final number of animals/blood sam-
ples collected per group, was not reported. Never-
theless, the results appear to have been sufficient

for statistical analysis, so I scored this metric as
medium.

Domain 5: Outcome Assessment

Continued on next page . ..

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.. . continued from previous page

Study Citation: Drew, RT; Patel, JM; Lin, FN (1978). Changes in serum enzymes in rats after inhalation of organic solvents singly and in combination

Toxicology and Applied Pharmacology, 45(3), 809-819
Data Type: 4-hour inhalation

HERO ID: 67913

Domain Metric Rating^ MWF* Score Comments^

Metric 16:

Outcome Assessment Methodology

Low

The outcome assessment methodology for this
acute exposure study was limited to clinical chem-
istry/biochemistry parameters, specifically, serum
enzyme analysis.

Metric 17:

Consistency of Outcome Assessment

High

The outcome assessment methodology appeared to
be consistent among the groups in terms of the
procedures used to measure the different serum en-
zymes. There was no indication that methods dif-
fered between groups for timing of blood collection
for analysis.

Metric 18:

Sampling Adequacy

High

Details regarding sampling for the outcome(s) of in-
terest were reported and acceptable for the outcomes
of interest.

Metric 19:

Blinding of Assessors

Not Rated

No subjective endpoints were evaluated in this study.

Metric 20:

Negative Control Response

High

Each rat served as its own control prior to exposure.

Domain 6: Confounding / Variable Control

Metric 21:

Confounding Variables in Test Design and

Low

There were no confounding differences reported

Procedures

among the study groups; however, initial body

weight or food/water intake were not reported. Ad-
ditionally, respiratory rate was not reported, but
1,4-dioxane is a potential respiratory irritant, so I
downgraded the score to low.

Metric 22:

Health Outcomes Unrelated to Exposure

Medium

Data on attrition and health outcomes unrelated to
exposure for each study group were not reported be-
cause only differences among groups for the evalu-
ated outcomes were noted.

Domain 7: Data Presentation and Analysis

Metric 23:

Statistical Methods

High

Statistical methods were described in sufficient de-

tail and were appropriate for the data sets.

Metric 24:

Reporting of Data

Low

Data presentation is incomplete. No data were pre-
sented for control groups.

Overall Quality Determination"1

Medium

2.2

Extracted Yes

Continued on next page . ..

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. continued from previous page

Study Citation: Drew, RT; Patel, JM; Lin, FN (1978). Changes in serum enzymes in rats after inhalation of organic solvents singly and in combination

Toxicology and Applied Pharmacology, 45(3), 809-819
Data Type:	4-hour inhalation

HERO ID:	67913

Domain	Metric	Rating^ MWF* Score	Comments^

* MWF = Metric Weighting Factor

t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

+ The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

if any metric is Unacceptable

Overall rating =

J]. (Metric Score; x MWF;) / J] . MWFj

(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 7 of 187

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Table 2: Animal toxicity evaluation results of Uno et al 1994 for an acute oral study on mechanistic (gene expression/omics,
genotoxicity) outcomes

Study Citation: Uno, Y; Takasawa, H; Miyagawa, M; Inoue, Y; Murata, T; Yoshikawa, K (1994). An in vivo-in vitro replicative DNA synthesis (RDS)
test using rat hepatocytes as an early prediction assay for nongenotoxic hepatocarcinogens screening of 22 known positives and 25
noncarcinogens Mutation Research, 320(3), 189-205
Data Type: Acute oral

HERO ID: 194385

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Test substance was identified definitively.

Metric 2:

Test Substance Source

Medium

The source of the test substance was reported
(Tokyo Chem Industry Co). A batch/lot number
was not reported.

Metric 3:

Test Substance Purity

Low

Purity was not reported.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

Low

A concurrent negative/vehicle control group was
tested but it appears that results for the control were
only based on T = 0, rather than a true control,
which was sampled at each time point (i.e., also 24,
39, 48 hours post-treatment/administration of vehi-
cle, i.e., see Table 1).

Metric 5:

Positive Controls

Not Rated

Metric 6:

Randomized Allocation

Low

The study authors did not report how animals were
allocated to study groups.

Domain 3: Exposure Characterization

Metric 7:

Preparation and Storage of Test Substance

Low

The test substance was dissolved or suspended in
corn oil; however, no other details were provided on
test substance preparation or storage methods.

Metric 8:

Consistency of Exposure Administration

High

Details of exposure were reported and there was
no indication to suggest that the exposures differed
among the groups.

Metric 9:

Reporting of Doses/Concentrations

Medium

The administered doses (1000 and 2000 mg/kg via
gavage) were reported. It appears that these were
per body weight doses, although not specifically
stated.

Metric 10:

Exposure Frequency and Duration

High

Exposure frequency and duration were reported (sin-
gle exposure with evaluation at up to 48 hours post-

exposure.. These appear acceptable for the intended
outcomes for the study (mechanistic).

Continued on next page . ..

Page 8 of 187

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.. . continued from previous page

HERO ID: 194385

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 11:

Number of Exposure Groups and Dose Spac-

High

The number of exposure groups and dose spacing

ing

were considered adequate to address the purpose of

llxO

the study and were justified by the study authors

(were based on the MTD).

Metric 12:

Exposure Route and Method

High

The exposure route and method were reported and

were considered appropriate for the purpose of the

study.

Domain 4: Test Organism

Metric 13:

Test Animal Characteristics

Medium

The test animal species, strain, age, sex, and source

were reported; however, body weight and health sta-

tus at the start of the study were not reported.

Metric 14:

Adequacy and Consistency of Animal Hus-

Medium

Most husbandry conditions (temperature and light)

bandry Conditions

were reported and were similar for all groups. Hu-

midity was not reported.

Metric 15:

Number per Group

Medium

The number per group (n = 4) was smaller than

is typical for a study of this type (acute exposure)

but was appropriate for the intended outcomes and

purpose of the study.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

Medium

The outcome assessment methodology was reported

and was sensitive for the outcomes of interest al-

though it's not clear that the duration (up to 48

hours post-exposure) was sufficient to address the

intended outcomes.

Metric 17:

Consistency of Outcome Assessment

High

The outcome assessment methodology appeared to

be consistent among the groups.

Metric 18:

Sampling Adequacy

Medium

Sampling methods appear to have been appropriate

for addressing the outcomes of interest (2000 hepa-

tocytes/liver (n = 4)) were evaluated for replicative

DNA synthesis (RDS). It's not clear, however, how

cell viability was determined (i.e., how many cells

were sampled).

Metric 19:

Blinding of Assessors

Not Rated

No subjective outcomes were evaluated in this study.

Metric 20:

Negative Control Response

High

Biological responses of the negative control group

were adequate.

Domain 6: Confounding / Variable Control

Metric 21:

Confounding Variables in Test Design and

Medium

No confounding variables in test design were re-

Procedures

ported; however, initial body weight and food/water

intake were not reported.

Continued on

next page . .

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.. . continued from previous page

Study Citation:

Data Type:
HERO ID:

Uno, Y; Takasawa, H; Miyagawa, M; Inoue, Y; Murata, T; Yoshikawa, K (1994). An in vivo-in vitro replicative DNA synthesis (RDS)
test using rat hepatocytes as an early prediction assay for nongenotoxic hepatocarcinogens screening of 22 known positives and 25
noncarcinogens Mutation Research, 320(3), 189-205
Acute oral
194385

Domain Metric

Rating^

MWF*

Score

Comments^

Metric 22: Health Outcomes Unrelated to Exposure

Medium

X 1

2

Data on attrition and health outcomes unrelated to
exposure for each study group were not reported be-
cause only differences among groups for the evalu-
ated outcomes were noted.

Domain 7: Data Presentation and Analysis









Metric 23: Statistical Methods

High

X 1

1

Statistical methods were reported and were appro-
priate for the data sets.

Metric 24: Reporting of Data

High

x 2

2

Data for exposure-related findings were presented
(RDS incidence and cell viability, only mechanistic
outcomes were reported).

Overall Quality Determination1"

Medium



1.8



Extracted

Yes







MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; X MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

This metric met the criteria for high confidence as expected for this type of study

Page 10 of 187

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Table 3: Animal toxicity evaluation results of Mattie et al 2012 for a 6-hour inhalation study on neurological/behavioral outcomes

Study Citation: Mattie, DR; Bucher, TW; Carter, AL; Stoffregen, DE; Reboulet, JE (2012). Acute inhalation toxicity study of 1, 4-dioxane in rats

(Rattus norvegicus) GRA and 1(20), 29
Data Type: 6-hour inhalation study - neuro

HERO ID: 3563367

Domain Metric Rating^ MWF* Score Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Clearly identified: 1,4-dioxane ((formula: C4H802);
CAS # 123-91-1)

Metric 2:

Test Substance Source

Medium

Purchased from Sigma-Aldrich, Inc.. (batch no. not
reported)

Metric 3:

Test Substance Purity

High

>99% purity

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

Concurrent negative controls were exposed to clean
air. 2 separate control groups were used to ensure
concurrent exposure group for all 5 exposure levels
(only 4 total exposure chambers).

Metric 5:

Positive Controls

Not Rated

Positive control not required for study type (OPPTS

870.1300)

Metric 6:

Randomized Allocation

High

Animals were "randomly selected for each exposure
group".

Domain 3: Exposure Characterization

Metric 7:

Preparation and Storage of Test Substance

High

Vapor generation method was adequately reported.

Metric 8:

Consistency of Exposure Administration

Medium

Exposure methods were consistent between groups.
In the low-dose group (target 100 ppm), there was a
problem in the air handling system of the chamber,
resulting in a large spike in concentration during the
first hour. The issue was resolved, but resulted in a
large standard deviation.

Metric 9:

Reporting of Doses/Concentrations

High

Target, nominal, and analytical concentrations re-
ported (Table 3). Exposure chamber concentrations
were continuously sampled and the concentration
determined approximately every 40 seconds by FTIR
analysis for each entire 6 hour exposure.

Metric 10:

Exposure Frequency and Duration

High

Exposure duration consistent with cited guideline
(OPPTS 870.1300)

Metric 11:

Number of Exposure Groups and Dose Spac-

High

Five exposure groups plus concurrent controls were

ing

used. Exposure levels were based on levels in previ-

ous studies.

Continued on next page . ..

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. continued from previous page

Study Citation: Mattie, DR; Bucher, TW; Carter, AL; Stoffregen, DE; Reboulet, JE (2012). Acute inhalation toxicity study of 1, 4-dioxane in rats

(Rattus norvegicus) GRA and 1(20), 29
Data Type: 6-hour inhalation study - neuro

HERO ID: 3563367

Domain

Metric

Rating^ MWF* Score

Comments^

Metric 12: Exposure Route and Method

High

x 1

Dynamic, whole-body exposure with 15 complete
fresh air changes per hour; individually housed in
690 L chambers. Any aerosols that were formed dur-
ing vaporization process were captured by a patch of
glass wool upstream, so nose-only exposure was not
necessary.

Domain 4: Test Organism

Metric 13: Test Animal Characteristics

High

Metric 14: Adequacy and Consistency of Animal Hus- High
bandry Conditions

Metric 15: Number per Group High

X 2 2 Albino inbred Fischer (CDF®) [F344/DuCrl] rats.

Age not reported. Based on weights (150-200g for
males, 125-175g for females) they were young adults.

X 1 1 Husbandry conditions were the same between

groups. All animals acclimated to exposure cham-
bers for 5 days before exposure.

X 1 1 10/sex/group; 5/sex sacrificed two days after start

of exposure, 5/sex sacrificed 2 weeks after exposure
(minimum guideline: 5/sex/group observed for 14
days)

Domain 5: Outcome Assessment

Metric 16: Outcome Assessment Methodology

Metric 17: Consistency of Outcome Assessment
Metric 18: Sampling Adequacy

Metric 19: Blinding of Assessors
Metric 20: Negative Control Response

High

High
High

x 2

x 1
x 1

Unacceptable x 1

2 Clinical signs of neurotoxicity (autonomic effects,

central nervous system effects, and reactivity to han-
dling or sensory stimuli)

1 Assessment identical across groups.

1 Sampling consisted with cited guideline (OPPTS
870.1300)

4 No reporting of blinding status of examiners during

subjective assessments of clinical signs of neurotox-
icity.

4 Results of clinical signs evaluations not reported for

control or exposure group.

Domain 6: Confounding / Variable Control

Metric 21: Confounding Variables in Test Design and Low
Procedures

x 2

Methods section states that evaluations of respi-
ration were conducted, but respiratory rate was
not reported (no reporting of clinical signs, or lack
thereof). Rated as low since 1,4-dioxane is a respi-
ratory irritant.

Continued on next page . ..

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.. . continued from previous page

Study Citation: Mattie, DR; Bucher, TW; Carter, AL; Stoffregen, DE; Reboulet, JE (2012). Acute inhalation toxicity study of 1, 4-dioxane in rats

(Rattus norvegicus) GRA and 1(20), 29
Data Type:	6-hour inhalation study - neuro

HERO ID:	3563367

Domain

Metric

Rating^ MWF* Score

Comments^

Metric 22: Health Outcomes Unrelated to Exposure

Medium

x 1

No mortalities were reported. Minimal serous exu-
date and few acute and chronic leukocyte infiltrates
that were observed in a small number of rats dis-
tributed across all groups, controls and treated, were
attributed to "environment irritants and/or a mild
resolving bacterial infection"; observed at both 2 day
and 14 day sacrifice. This is not expected to impact
neurological assessment.

Domain 7: Data Presentation and Analysis







Metric 23: Statistical Methods

Unacceptable x 1

4

No mention of statistical analysis of clinical neuro-
toxicity evaluation (data not reported).

Metric 24: Reporting of Data

Unacceptable x 2

8

Results of clinical signs evaluations not reported for
control or exposure group.

Overall Quality Determination1"

Unacceptable**

1.7



Extracted

No







* Consistent with our Application of Systematic Review in TSCARisk Evaluations document, if a metric for a data source receives a score of Unacceptable (score = 4),
EPA will determine the study to be unacceptable. In this case, one or more of the metrics were rated as unacceptable. As such, the study is considered unacceptable and
the score is presented solely to increase transparency.

MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; X MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 13 of 187

-------
Table 4: Animal toxicity evaluation results of Mattie et al 2012 for a 6-hour inhalation systemic effects study on hepatic, renal,
irritation, respiratory, body weight outcomes

Study Citation: Mattie, DR; Bucher, TW; Carter, AL; Stoffregen, DE; Reboulet, JE (2012). Acute inhalation toxicity study of 1, 4-dioxane in rats

(Rattus norvegicus) GRA and 1(20), 29
Data Type: 6-hour inhalation study - systemic effects

HERO ID: 3563367

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Clearly identified: 1,4-dioxane ((formula: C4H802);
CAS # 123-91-1)

Metric 2:

Test Substance Source

Medium

Purchased from Sigma-Aldrich, Inc.. (batch no. not
reported)

Metric 3:

Test Substance Purity

High

>99% purity

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

Concurrent negative controls were exposed to clean
air. 2 separate control groups were used to ensure
concurrent exposure group for all 5 exposure levels
(only 4 total exposure chambers).

Metric 5:

Positive Controls

Not Rated

Positive control not required for study type (OPPTS

870.1300)

Metric 6:

Randomized Allocation

High

Animals were "randomly selected for each exposure
group".

Domain 3: Exposure Characterization

Metric 7:

Preparation and Storage of Test Substance

High

Vapor generation method was adequately reported.

Metric 8:

Consistency of Exposure Administration

Medium

Metric 9:

Reporting of Doses/Concentrations

High

Metric 10:

Exposure Frequency and Duration

High

Exposure duration consistent with cited guideline
(OPPTS 870.1300)

Metric 11:

Number of Exposure Groups and Dose Spac-

High

Five exposure groups plus concurrent controls were

ing

used. Exposure levels were based on levels in previ-

ous studies.

Continued on next page . ..

Page 14 of 187

-------
. continued from previous page

Study Citation: Mattie, DR; Bucher, TW; Carter, AL; Stoffregen, DE; Reboulet, JE (2012). Acute inhalation toxicity study of 1, 4-dioxane in rats

(Rattus norvegicus) GRA and 1(20), 29
Data Type: 6-hour inhalation study - systemic effects

HERO ID: 3563367

Domain

Metric

Rating^ MWF* Score

Comments^

Metric 12: Exposure Route and Method

High

x 1

Domain 4: Test Organism

Metric 13: Test Animal Characteristics

High

Metric 14: Adequacy and Consistency of Animal Hus- High
bandry Conditions

Metric 15: Number per Group High

X 2 2 Albino inbred Fischer (CDF®) [F344/DuCrl] rats.

Age not reported. Based on weights (150-200g for
males, 125-175g for females) they were young adults.

X 1 1 Husbandry conditions were the same between

groups. All animals acclimated to exposure cham-
bers for 5 days before exposure.

X 1 1 10/sex/group; 5/sex sacrificed two days after start

of exposure, 5/sex sacrificed 2 weeks after exposure
(minimum guideline: 5/sex/group observed for 14
days)

Domain 5: Outcome Assessment

Metric 16: Outcome Assessment Methodology

Metric 17: Consistency of Outcome Assessment
Metric 18: Sampling Adequacy

Metric 19: Blinding of Assessors

Metric 20: Negative Control Response

High X 2 2 Hepatic, Renal - OW, HP

Respiratory - HP of entire respiratory tract, includ-
ing nasal sections

Body weight - at randomization, prior to exposure,
weekly during post-exposure, and at necropsy

High X 1 1 Assessment identical across groups.

High X 1 1 Sampling consisted with cited guideline (OPPTS

870.1300)

Not Rated NA NA Only non-subjective outcomes and initial

histopathological evaluations performed; blind-
ing not necessary.

Medium X 1 2 Control histopathological data were not explicitly

stated, but based on qualitative statements regard-
ing what was found in higher exposure groups, it is
inferred that lesions were not observed in controls.
Qualitative statement regarding no statistically sig-
nificant changes in organ weight or body weight cov-
ers both control and exposure groups.

Domain 6: Confounding / Variable Control

Continued on next page . .

Page 15 of 187

-------
... continued from previous page

Study Citation: Mattie, DR; Bucher, TW; Carter, AL; Stoffregen, DE; Reboulet, JE (2012). Acute inhalation toxicity study of 1, 4-dioxane in rats

(Rattus norvegicus) GRA and 1(20), 29
Data Type:	6-hour inhalation study - systemic effects

HERO ID:	3563367

Domain	Metric	Rating^ MWF* Score	Comments^

Metric 21: Confounding Variables in Test Design and

Low

x 2

6

Methods section states that evaluations of respi-

Procedures







ration were conducted, but respiratory rate was







not reported (no reporting of clinical signs, or lack
thereof). Rated as low since 1,4-dioxane is a respi-
ratory irritant.

Metric 22: Health Outcomes Unrelated to Exposure

Medium

x 1

2

No mortalities were reported. Minimal serous exu-
date and few acute and chronic leukocyte infiltrates
that were observed in a small number of rats dis-
tributed across all groups, controls and treated, were
attributed to "environment irritants and/or a mild
resolving bacterial infection"; observed at both 2 day
and 14 day sacrifice.

Domain 7: Data Presentation and Analysis









Metric 23: Statistical Methods

Medium

x 1

2

BW and OW data analyzed by t-test and ANOVA.
No statistical analysis of lesion incidence. Exposure-
related nasal lesion incidence is reported in higher
exposure groups - if it is assumed that lesion inci-
dence is 0/5 for groups without explicitly reported
lesions, statistical analysis could be conducted . In-
cidental findings that were observed in "all groups"
were reported qualitatively only (not adequate for
statistical analysis).

Metric 24: Reporting of Data

Medium

x 2

4

BW/OW - Qualitative (no effects)

Histo - Exposure-related nasal lesion incidence is re-
ported in higher exposure groups (assumed 0/5 for
other groups, but not explicitly reported). Inciden-
tal findings that were observed in "all groups" were
reported qualitatively only.

Overall Quality Determination"1'

High —i

¦ Medium^





Extracted

Yes







MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

if any metric is Unacceptable

Overall rating =

J]. (Metric Score; x MWF;) / J] . MWFj

(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

§ Evaluator's explanation for rating change: "Due to some limitations in data reporting (requiring reader to make inferences) and study author's indication that other
environmental irritants or infection may have been present, the study was downgraded to medium. However, since nasal lesions were observed at high exposure levels
(in addition to the nasal irritation findings in all groups), the study still appears adequate to identify exposure-related findings."

Page 16 of 187

-------
Table 5: Animal toxicity evaluation results of Dow et al 1989 for a single dose in vivo DNA synthesis study on hepatic, genotoxicity,
body weight outcomes

Study Citation: Dow Chemical Company (1989). Differentiation of the mechanisms of oncogenicity of 1,4-dioxane and 1,3-hexachlorobutadiene in the
rat

Data Type:	Single dose in vivo DNA synthesis

HERO ID:	4158030

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance













Metric 1:

Test Substance Identity

High

X

2

2

1,4-dioxane

Metric 2:

Test Substance Source

Medium

X

1

2

Baker Chemical Company; no batch number, but













purity was analyzed by study laboratory

Metric 3:

Test Substance Purity

High

X

1

1

>99%

Domain 2: Test Design













Metric 4:

Negative and Vehicle Controls

High

X

2

2

Concurrent vehicle (saline) control was used

Metric 5:

Positive Controls

Unacceptable

X

1

4

No positive control; in vivo genotoxicity study de-













sign indicates one should have been used (DMN was













used in the repeat dose study only)

Metric 6:

Randomized Allocation

High

X

1

1

Animals were computer randomized into treatment













groups in all experiments

Domain 3: Exposure Characterization











Metric 7:

Preparation and Storage of Test Substance

High

X

1

1

Storage details not reported. Mixed with saline for













gavage administration.

Metric 8:

Consistency of Exposure Administration

High

X

1

1

Exposure conditions consistent between groups.

Metric 9:

Reporting of Doses/Concentrations

High

X

2



Replicate 1: 0, 100, or 1000 mg/kg













Replicate 2: 0, 10, 100, or 1000 mg/kg

Metric 10:

Exposure Frequency and Duration

High

X

1

1

Once, sacrificed after 7 d

Metric 11:

Number of Exposure Groups and Dose Spac-

High

X

1

1

2-3 doses plus negative control (two replicates)

Metric 12:

ing

Exposure Route and Method

Medium

X

1

2

No rationale was provided for switching from gav-













age (this study) to repeat-dose drinking water study

(accompanying study). Other compounds (HCBD,
DMN) were administered via gavage for both stud-
ies. However, BWG was decreased by —45-55% fol-
lowing single gavage administration of 1000 mg/kg;
this BW effect was not observed with drinking wa-
ter administration of 1000 mg/kg over 11 weeks. SO
perhaps the change in route was due to the decreased
body weight associated with gavage administration.

Domain 4: Test Organism

Metric 13:

Test Animal Characteristics High

x 2

2

Male SD rats (Spartan Research). Based on weight
(180-260g), they were adult animals.

Continued on next page . ..

Page 17 of 187

-------
.. . continued from previous page

Study Citation: Dow Chemical Company (1989). Differentiation of the mechanisms of oncogenicity of 1,4-dioxane and 1,3-hexachlorobutadiene in the
rat

Data Type: Single dose in vivo DNA synthesis

HERO ID: 4158030

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 14:

Adequacy and Consistency of Animal Hus-
bandry Conditions

Medium

Husbandry was consistent between groups (wire
cages, environmentally controlled rooms, food and
water ad libitum). Number of rats/cage was not re-
ported, environmental conditions not reported.

Metric 15:

Number per Group

High

4/group

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

Genotox, organ weight, and histology of liver (cancer
target organ); body weight and food consumption
also monitored.

Metric 17:

Consistency of Outcome Assessment

High

Consistent evaluation across study groups

Metric 18:

Sampling Adequacy

High

4/group

Metric 19:

Blinding of Assessors

Not Rated

Only non-subjective and initial histological evalua-
tions; blinding not required.

Metric 20:

Negative Control Response

High

negative control response was reported; no devia-
tions from normal were reported.

Domain 6: Confounding / Variable Control

Metric 21:

Confounding Variables in Test Design and
Procedures

Unacceptable

Initial BW 180-260 g (not reported per group).
Body weight gains decreased 45-55% at 1000 mg/kg
and 33-40% at 10-100 mg/kg. Decreased food con-
sumption (magnitude not reported) associated with
decreased BW. This may be the reason that drink-
ing water route was used for repeat-dose study (same
high exposure dose level).

Metric 22:

Health Outcomes Unrelated to Exposure

Low

Weight loss may have been due to exposure route
(bolus exposure) as opposed to (or in addition to)
toxic effects. No weight effects observed at the same
exposure level in accompanying repeated exposure
drinking water study.

Domain 7: Data Presentation and Analysis

Metric 23:

Statistical Methods

Medium

Continuous data were compared by Dunnett's t-test.
No statistical analysis of histopathological findings.
Histological findings only reported qualitatively.

Metric 24:

Reporting of Data

Medium

DNA synthesis, liver weight, and BWG reported
quantitatively with statistics. Histopathological
findings reported qualitatively (present or absent at

dose).

Overall Quality Determination1"

Unacceptable**

1.6

Extracted

Continued on next page . ..

Page 18 of 187

-------
. continued from previous page

Study Citation: Dow Chemical Company (1989). Differentiation of the mechanisms of oncogenicity of 1,4-dioxane and 1,3-hexachlorobutadiene in the
rat

Data Type:	Single dose in vivo DNA synthesis

HERO ID:	4158030

Domain	Metric	Rating^ MWF* Score	Comments^

** Consistent with our Application of Systematic Review in TSCARisk Evaluations document, if a metric for a data source receives a score of Unacceptable (score = 4),
EPA will determine the study to be unacceptable. In this case, one or more of the metrics were rated as unacceptable. As such, the study is considered unacceptable and
the score is presented solely to increase transparency.

* MWF = Metric Weighting Factor

t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

+ The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

if any metric is Unacceptable

Overall rating =

J]. (Metric Score; x MWF;) / J] . MWFj

(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 19 of 187

-------
2 Short-term (1-30 days)

Table 6: Animal toxicity evaluation results of Goldberg et al 1964 for a 10-day inhalation study on neurological/behavior, body
weight outcomes

Study Citation: Goldberg, ME; Johnson, HE; Pozzani, UC; Smyth, HF, Jr (1964). Effect of repeated inhalation of vapors of industrial solvents on
animal behavior: I. Evaluation of nine solvent vapors on pole-climb performance in rats American Industrial Hygiene Association
Journal, 25(4), 369-375
Data Type: 10-day inhalation

HERO ID: 58035

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

x 2

Test substance was identified definitively.

Metric 2:

Test Substance Source

Low

x 1

The report states that chemicals were obtained com-
mercially; however, source or analytical verification
of test substance were not reported. No batch/lot
numbers were reported. The omitted details are not
likely to have a substantial impact on results.

Metric 3:

Test Substance Purity

Low

x 1

Purity and grade were not reported.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

x 2

A concurrent negative control group was tested and
was appropriate.

Metric 5:

Positive Controls

Not Rated

A concurrent positive control group is not necessary
for this study type.

Metric 6:

Randomized Allocation

High

x 1

Animals were randomized and distributed into
groups.

Domain 3: Exposure Characterization

Metric 7:

Preparation and Storage of Test Substance

Medium

x 1

Methods and equipment used for generating the test
atmospheres were reported; however, storage condi-
tions for the test substance were not reported, so I
downgraded the score for this metric to medium.

Metric 8:

Consistency of Exposure Administration

High

X 1

Details of the exposure administration were reported
and exposures were administered consistently across
study groups.

Metric 9:

Reporting of Doses/Concentrations

Low

x 2

Actual concentrations were not reported. Concen-
trations were reported as nominal values. Vapor test
concentrations were monitored during the exposures
and air flows were adjusted so that the actual vapor

concentrations were within 10% of nominal concen-
trations. Due to the lack of reporting of actual con-
centrations for vapor exposures, I downgraded this
metric to low.

Continued on next page . ..

Page 20 of 187

-------
.. . continued from previous page

HERO ID: 58035

Domain Metric Rating^ MWF* Score Comments^

Metric 10:

Exposure Frequency and Duration

High

The exposure frequency and duration of exposure
were reported and were appropriate for this study
type and the outcomes of interest.

Metric 11:

Number of Exposure Groups and Dose Spac-

High

The number of exposure groups and

ing

dose/concentration spacing were adequate to

address the purpose of the study. Selected concen-
trations were not justified by the study authors but
the range of concentrations was appropriate.

Metric 12:

Exposure Route and Method

Low

The route of exposure (inhalation) was reported and
was suited to the test substance. The method of
exposure was not specifically stated, but appears
to have been dynamic whole-body exposure, based
on the study methods description, and is considered
suitable for the test substance. The number of air
changes per hour was not reported, so I downgraded
the score to low.

Domain 4: Test Organism

Metric 13:

Test Animal Characteristics

Medium

The test animal species, strain, sex, age, and start-
ing body weight were reported. Health status at the
start of the study was not reported.

Metric 14:

Adequacy and Consistency of Animal Hus-

Low

Husbandry conditions (temperature, humidity, light

bandry Conditions

cycle) were not sufficiently reported to evaluate if

husbandry was adequate and similar among the
groups, so I downgraded the score for this metric
to low.

Metric 15:

Number per Group

Medium

The number of animals per study group (8/group)
was lower than the typical number used in repeated-
dose studies, but sufficient for statistical analysis
and this minor limitation is unlikely to have a sub-
stantial impact on results.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

Medium

The outcome assessment methodology was reported
and specific for the outcomes of interest (neurobe-
havioral effects). However, the study did not include
a post-mortem examination of neural tissue.

Continued on

next page .

Page 21 of 187

-------
.. . continued from previous page

HERO ID: 58035

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 17:

Consistency of Outcome Assessment

Low

Outcome assessments were not adequately reported
to allow a determination of whether evaluations
were performed consistently. The report states that
tests made from zero to two hours after exposure
gave maximal effects, and results were reported as
the quantal response at the time of maximum ef-
fect; however, not all time points evaluated were re-
ported.

Metric 18:

Sampling Adequacy

Low

Details regarding sampling were not reported to de-
termine if sampling was adequate for all groups. For
example, it's not stated how many of the eight an-
imals per group were evaluated, neither in the text
nor in the results table (Table IV).

Metric 19:

Blinding of Assessors

Low

Blinding status was not reported in this study. Neu-
robehavioral assessments typically need to be con-
ducted by blinded assessors, however, there was a
quantitative aspect to the assessment (i.e., response
time). While blinding would have been preferred,
it is not as crucial in this case as it is for purely
subjective observations.

Metric 20:

Negative Control Response

Low

Negative control data were not shown for all out-
comes; however, negative control data were com-
pared to treatment groups for purposes of determin-
ing effects on evaluated outcomes (e.g., body weight,
avoidance response, escape response, as shown in Ta-
ble IV). These uncertainties are unlikely to have a
substantial impact on results.

Domain 6: Confounding / Variable Control

Metric 21:

Confounding Variables in Test Design and

Low

There were no confounding differences reported

Procedures

among the study groups; however, initial body

weight or food/water intake were not reported. Ad-
ditionally, respiratory rate was not reported, but
1,4-dioxane is a potential respiratory irritant, so I
scored this metric as low.

Metric 22:

Health Outcomes Unrelated to Exposure

Medium

Data on attrition and health outcomes unrelated to
exposure for each study group were not reported
because only substantial differences among groups
were noted.

Domain 7: Data Presentation and Analysis

Continued on next page . ..

Page 22 of 187

-------
.. . continued from previous page

Study Citation:

Data Type:
HERO ID:

Goldberg, ME; Johnson, HE; Pozzani, UC; Smyth, HF, Jr (1964). Effect of repeated inhalation of vapors of industrial solvents on
animal behavior: I. Evaluation of nine solvent vapors on pole-climb performance in rats American Industrial Hygiene Association
Journal, 25(4), 369-375
10-day inhalation
58035

Domain Metric

Rating^

MWF*

Score

Comments^

Metric 23: Statistical Methods
Metric 24: Reporting of Data

Low
Low

X 1

x 2

3
6

Statistical methods were reported for body weight
data, but not for evaluation of avoidance and escape
response data. Mean values with standard devia-
tions were not reported for avoidance and escape re-
sponse data, so an independent analysis would not
be possible.

Body weight effects were reported (e.g., Table IV)
but data were not shown in full. Neurologi-
cal/behavioral effects, as reported in Table IV, were
observed, but data were not reported completely
(only %'s affected are shown).

Overall Quality Determination1"

Medium



2.2



Extracted

Yes









MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; X MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

This metric met the criteria for high confidence as expected for this type of study

Page 23 of 187

-------
Table 7: Animal toxicity evaluation results of Roy et al 2005 for an in vivo micronucleus assay - main study on genotoxicity outcomes

Study Citation: Roy, SK; Thilagar, AK; Eastmond, DA (2005). Chromosome breakage is primarily responsible for the micronuclei induced by 1,4-

dioxane in the bone marrow and liver of young CD-I mice Mutation Research, 586(1,1), 28-37
Data Type: In vivo micronucleus assay - main study

HERO ID: 196094

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

1,4-Dioxane (CAS No. 123-91-1)

Metric 2:

Test Substance Source

Medium

1,4-Dioxane (99.9%, HPLC grade) was obtained
from Aldrich Chemical Company (Milwaukee, WI).
Batch ^ not reported, no independent analytical
verification.

Metric 3:

Test Substance Purity

High

99.9%, HPLC grade

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

The negative control group was administered 0.9%
NaCl via gavage.

Metric 5:

Positive Controls

High

Animals in the positive control group were in-

jected intraperitoneally with vinblastine sulfate
(0.85 mg/kg per day).

Metric 6:

Randomized Allocation

Low

The study did not report how animals were placed
into groups.

Domain 3: Exposure Characterization

Metric 7:

Preparation and Storage of Test Substance

Low

Storage and preparation were not reported explic-
itly; based on methods, it appears animals were gav-
aged with undiluted test substance.

Metric 8:

Consistency of Exposure Administration

Medium

Exposure similar between negative control and expo-
sure groups; gavage volume was not reported. Posi-
tive control i.p. injection.

Metric 9:

Reporting of Doses/Concentrations

High

Gavage doses reported.

Metric 10:

Exposure Frequency and Duration

High

gavage, once daily for 5 days.

Metric 11:

Number of Exposure Groups and Dose Spac-
ing

High

Three exposure groups, plus controls. Doses selected
based on range-finding study.

Metric 12:

Exposure Route and Method

High

Gavage

Domain 4: Test Organism

Metric 13:

Test Animal Characteristics

High

Young 21-day-old male CD-I mice were purchased
from Harlan (Indianapolis, Indiana, USA).

Metric 14:

Adequacy and Consistency of Animal Hus-
bandry Conditions

High

Conditions were similar between groups and consis-
tent with standard practices.

Metric 15:

Number per Group

High

5/group; appropriate for subacute exposure study

Domain 5: Outcome Assessment

Continued on next page . ..

Page 24 of 187

-------
... continued from previous page

Study Citation: Roy, SK; Thilagar, AK; Eastmond, DA (2005). Chromosome breakage is primarily responsible for the micronuclei induced by 1,4-

dioxane in the bone marrow and liver of young CD-I mice Mutation Research, 586(1,1), 28-37
Data Type:	In vivo micronucleus assay - main study

HERO ID:	196094

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 16:

Outcome Assessment Methodology

High

X

2

2

Micronuclei evaluation in bone marrow and hepatic













cells; hepatic cells also evaluated for proliferation













(BrdU) to evaluation potential origins of micronu-

Metric 17:

Consistency of Outcome Assessment

High

X

1

1

Animals evaluated the same across groups.

Metric 18:

Sampling Adequacy

High

X

1

1



Metric 19:

Blinding of Assessors

High

X

1

1

Slides were randomized and coded prior to scoring.

Metric 20:

Negative Control Response

High

X

1

1

Control responses reported, no deviation from ex-













pected reported. Expected results observed in posi-













tive controls.

Domain 6: Confounding / Variable Control











Metric 21:

Confounding Variables in Test Design and

Medium

X

2

4

The lack of reporting of initial body weights and



Procedures









food/water intake are unlikely to have an impact











on results due to subacute duration and endpoints













assessed.

Metric 22:

Health Outcomes Unrelated to Exposure

High

X

1

1

No indication of attrition or health outcomes unre-











lated to exposure.

Domain 7: Data Presentation and Analysis











Metric 23:

Statistical Methods

High

X

1

1

ANOVA, regression analysis on transformed data;











post-hoc Fisher's protected least significant differ-













ence. Critical values were determined using a 0.05













probability of type
I error.

Metric 24:

Reporting of Data

High

X

2

2

Data presented quantitatively in tables or figures.

Overall Quality Determination1"

High

1.3

Extracted

Yes





MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; x MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 25 of 187

-------
Table 8: Animal toxicity evaluation results of Roy et al 2005 for an in vivo micronucleus assay - range-finding study on genotoxicity,
mortality outcomes

Study Citation: Roy, SK; Thilagar, AK; Eastmond, DA (2005). Chromosome breakage is primarily responsible for the micronuclei induced by 1,4-

dioxane in the bone marrow and liver of young CD-I mice Mutation Research, 586(1,1), 28-37
Data Type: In vivo micronucleus assay - range-finding study

HERO ID: 196094

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

x 2

1,4-Dioxane (CAS No. 123-91-1)

Metric 2:

Test Substance Source

Medium

x 1

1,4-Dioxane (99.9%, HPLC grade) was obtained
from Aldrich Chemical Company (Milwaukee, WI).
Batch ^ not reported, no independent analytical
verification.

Metric 3:

Test Substance Purity

High

x 1

99.9%, HPLC grade

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

x 2

Methods states: "an initial range-finding study with
three animals per dose was performed at doses rang-
ing from 250 to 5000 mg/kg bw"., but results indi-
cate a control group (0 mg/kg-day). The main study
dosed controls with 0.9% NaCl via gavage.

Metric 5:

Positive Controls

Not Rated

It doesn't appear that the range-finding study used

Metric 6: Randomized Allocation

Low

x 1

a positive control based on methods and results sec-
tions. However, the main study group used a pos-
itive control group were injected intraperitoneally
with vinblastine sulfate (0.85 mg/kg per day), and
saw expected results. Therefore, study design was
validated in the lab. For dose-range finding stud-
ies, it is likely OK not to have positive control, so I
selected N/A.

The study did not report how animals were placed
into groups.

Domain 3: Exposure Characterization

Metric 7: Preparation and Storage of Test Substance Low

Metric 8: Consistency of Exposure Administration Medium

Metric 9: Reporting of Doses/Concentrations Medium

X 1 3 Storage and preparation were not reported explic-

itly; based on methods, it appears animals were gav-
aged with undiluted test substance.

X 1 2 Exposure similar between negative control and ex-

posure groups; gavage volume was not reported. .

X 2 4 Undefined number of exposure groups from 250

mg/kg to 5000 mg/kg; mortality observed at >3500
mg/kg. Based on results section, there were 9 dose
groups between 250 and 3500 mg/kg, plus a nega-
tive control. Unclear if there were any dose groups
between 3500 mg/kg and 5000 mg/kg.

Continued on next page

Page 26 of 187

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.. . continued from previous page

Study Citation: Roy, SK; Thilagar, AK; Eastmond, DA (2005). Chromosome breakage is primarily responsible for the micronuclei induced by 1,4-

dioxane in the bone marrow and liver of young CD-I mice Mutation Research, 586(1,1), 28-37
Data Type: In vivo micronucleus assay - range-finding study

HERO ID: 196094

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 10

Exposure Frequency and Duration

Low

gavage, assumed once based on reporting and units

(mg/kg not mg/kg-day) . Not explicitly stated.

Metric 11

Number of Exposure Groups and Dose Spac-

High

At least 9 dose groups plus control.

Metric 12

ing

Exposure Route and Method

High

Gavage

Domain 4: Test Organism

Metric 13

Test Animal Characteristics

High

Young 21-day-old male CD-I mice were purchased

from Harlan (Indianapolis, Indiana, USA).

Metric 14

Adequacy and Consistency of Animal Hus-

High

Conditions were similar between groups and consis-

bandry Conditions

tent with standard practices.

Metric 15

Number per Group

High

3/group; adequate for range-finding

Domain 5: Outcome Assessment

Metric 16

Outcome Assessment Methodology

High

Micronuclei evaluation in bone marrow, mortality

Metric 17

Consistency of Outcome Assessment

High

Animals evaluated the same across groups.

Metric 18

Sampling Adequacy

High

Metric 19

Blinding of Assessors

High

Slides were randomized and coded prior to scoring.

Metric 20

Negative Control Response

High

Control responses reported, no deviation from ex-

pected reported.

Domain 6: Confounding / Variable Control

Metric 21

Confounding Variables in Test Design and

Medium

The lack of reporting of initial body weights and

Procedures

food/water intake are unlikely to have an impact

on results due to subacute duration and endpoints

assessed.

Metric 22

Health Outcomes Unrelated to Exposure

High

No indication of attrition or health outcomes unre-

lated to exposure.

Domain 7: Data Presentation and Analysis

Metric 23

Statistical Methods

Unacceptable

Unclear if statistics were conducted in range-finding

study (main study used ANOVA, regression analy-

sis on transformed data; post-hoc Fisher's protected

least significant difference). Data reporting of % mi-

cronucleus frequency inadequate for statistical anal-
ysis (no SD/SEM data).

Metric 24: Reporting of Data Unacceptable X 2 8 Micronuclei frequency reported as % only (no

SD/SEM data); mortality data reported qualita-
tively only ("some" mortality observed at >3500
mg/kg; unclear if there were doses between 3500 and
5000 mg/kg).

Continued on next page ...

Page 27 of 187

-------
... continued from previous page

Study Citation: Roy, SK; Thilagar, AK; Eastmond, DA (2005). Chromosome breakage is primarily responsible for the micronuclei induced by 1,4-

dioxane in the bone marrow and liver of young CD-I mice Mutation Research, 586(1,1), 28-37
Data Type:	In vivo micronucleus assay - range-finding study

HERO ID:	196094

Domain

Metric

Rating^ MWF* Score

Comments^

Overall Quality Determination1"



Unacceptable** 1.7



Extracted



No





* Consistent with our Application of Systematic Review in TSCARisk Evaluations document, if a metric for a data source receives a score of Unacceptable (score = 4),
EPA will determine the study to be unacceptable. In this case, one or more of the metrics were rated as unacceptable. As such, the study is considered unacceptable and
the score is presented solely to increase transparency.

MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; X MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 28 of 187

-------
Table 9: Animal toxicity evaluation results of Mattie et al 2012 for a 2-week inhalation study on neurological/behavioral, body
weight outcomes

Study Citation: Mattie, DR; Bucher, TW; Carter, AL; Stoffregen, DE; Reboulet, JE (2012). Acute inhalation toxicity study of 1, 4-dioxane in rats

(Rattus norvegicus) GRA and 1(20), 29
Data Type: 2-wk inhalation study - Neuro and BW

HERO ID: 3563367

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

x 2

Clearly identified: 1,4-dioxane ((formula: C4H802);

CAS # 123-91-1)

Metric 2:

Test Substance Source

Medium

x 1

Purchased from Sigma-Aldrich, Inc.. (batch no. not

reported)

Metric 3:

Test Substance Purity

High

x 1

>99% purity

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

x 2

Concurrent negative controls were exposed to clean

Metric 5:

Positive Controls

Not Rated

Positive control not required for study type (OECD

412)

Metric 6:

Randomized Allocation

High

x 1

Animals were "randomly selected for each exposure

group".

Domain 3: Exposure Characterization

Metric

Preparation and Storage of Test Substance

High

Metric

Consistency of Exposure Administration

High

Metric

Reporting of Doses/Concentrations

High

Metric

10:

Exposure Frequency and Duration

High

Metric

11:

Number of Exposure Groups and Dose Spac-

High

Vapor generation method was adequately reported.
Exposure methods were consistent between groups.
Target and analytical concentrations reported (Ta-
ble 4). Exposure chamber concentrations were con-
tinuously sampled and the concentration determined
approximately every 40 seconds by FTIR analysis for
each entire 6 hour exposure.

Exposure duration consistent with cited guideline
(OECD 412)

Three exposure groups plus concurrent controls were
used (consistent with guideline (OECD 412).. Meth-
ods section states that exposure levels were based
on levels in the accompanying acute (6-hr) study).
However, the discussion states that based on a gen-
eral lack of findings in acute study, the exposure
levels were based on the Kasai et al. (2008) 13-wk
study. Doses selected showed dose-response findings,
and are considered appropriate.

Continued on next page

Page 29 of 187

-------
. continued from previous page

Study Citation: Mattie, DR; Bucher, TW; Carter, AL; Stoffregen, DE; Reboulet, JE (2012). Acute inhalation toxicity study of 1, 4-dioxane in rats

(Rattus norvegicus) GRA and 1(20), 29
Data Type: 2-wk inhalation study - Neuro and BW

HERO ID: 3563367

Domain

Metric

Rating^ MWF* Score

Comments^

Metric 12: Exposure Route and Method

High

x 1

Domain 4: Test Organism

Metric 13: Test Animal Characteristics

High

Metric 14: Adequacy and Consistency of Animal Hus- High
bandry Conditions

Metric 15: Number per Group High

X 2 2 Albino inbred Fischer (CDF®) [F344/DuCrl] rats.

Age not reported. Based on weights (150-200g for
males, 125-175g for females) they were young adults.

X 1 1 Husbandry conditions were the same between

groups. All animals acclimated to exposure cham-
bers for 5 days before exposure.

X 1 1 16/sex/group; 8/sex sacrificed at end of exposure,

8/sex sacrificed 2 weeks after exposure (minimum
guideline: 5/sex/group per sacrifice)

Domain 5: Outcome Assessment

Metric 16: Outcome Assessment Methodology

High

x 2

Metric 17
Metric 18
Metric 19

Consistency of Outcome Assessment
Sampling Adequacy
Blinding of Assessors

Metric 20: Negative Control Response

High x 1

Unacceptable x 1

2 Body weight- at randomization, before each expo-

sure, weekly during recovery, at necropsy
Clinical signs of neurotoxicity (autonomic effects,
central nervous system effects, and reactivity to han-
dling or sensory stimuli)

1 Assessment identical across groups.

1 Sampling consisted with cited guideline (OECD 412)

4 No reporting of blinding status of examiners during

subjective assessments of clinical signs of neurotox-
icity.

4 Body weights and results of clinical signs evaluations

were not reported for control or exposure group.

Domain 6: Confounding / Variable Control

Metric 21: Confounding Variables in Test Design and Low
Procedures

Metric 22: Health Outcomes Unrelated to Exposure

Medium

x 2

x 1

No mortalities were reported. Unlike Acute study,
no mention of potential environmental irritants or
infection. Because those confounders were reported
in the acute study (and not specifically addressed in
subacute study), I rated as medium.

Continued on next page . ..

Page 30 of 187

-------
. continued from previous page

Study Citation: Mattie, DR; Bucher, TW; Carter, AL; Stoffregen, DE; Reboulet, JE (2012). Acute inhalation toxicity study of 1, 4-dioxane in rats

(Rattus norvegicus) GRA and 1(20), 29
Data Type:	2-wk inhalation study - Neuro and BW

HERO ID:	3563367

Domain Metric

Rating^

MWF*

Score

Comments^

Domain 7: Data Presentation and Analysis









Metric 23: Statistical Methods

Unacceptable

X 1

4

No mention of statistical analysis of clinical neuro-
toxicity evaluation (data not reported). Body weight
was reportedly analyzed with Student's t-test and
ANOVA (data not reported)

Metric 24: Reporting of Data

Unacceptable

x 2

8

Body weights and results of clinical signs evaluations
were not reported for control or exposure groups.

Overall Quality Determination"1'

Unacceptable**



1.7



Extracted

No









* Consistent with our Application of Systematic Review in TSCARisk Evaluations document, if a metric for a data source receives a score of Unacceptable (score = 4),
EPA will determine the study to be unacceptable. In this case, one or more of the metrics were rated as unacceptable. As such, the study is considered unacceptable and
the score is presented solely to increase transparency.

MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; X MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating,
ft This metric met the criteria for high confidence as expected for this type of study

Page 31 of 187

-------
Table 10: Animal toxicity evaluation results of Mattie et al 2012 for a 2-week inhalation systemic effects study on hepatic, renal,
irritation, respiratory, hematological and clinical chemistry outcomes

Study Citation: Mattie, DR; Bucher, TW; Carter, AL; Stoffregen, DE; Reboulet, JE (2012). Acute inhalation toxicity study of 1, 4-dioxane in rats

(Rattus norvegicus) GRA and 1(20), 29
Data Type: 2-wk inhalation study - systemic effects

HERO ID: 3563367

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Clearly identified: 1,4-dioxane ((formula: C4H802);

CAS # 123-91-1)

Metric 2:

Test Substance Source

Medium

Purchased from Sigma-Aldrich, Inc.. (batch no. not

reported)

Metric 3:

Test Substance Purity

High

>99% purity

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

Concurrent negative controls were exposed to clean

Metric 5:

Positive Controls

Not Rated

Positive control not required for study type (OECD

412)

Metric 6:

Randomized Allocation

High

Animals were "randomly selected for each exposure

group".

Domain 3: Exposure Characterization

Metric 7:

Preparation and Storage of Test Substance

High

Vapor generation method was adequately reported.

Metric 8:

Consistency of Exposure Administration

High

Exposure methods were consistent between groups.

Metric 9:

Reporting of Doses/Concentrations

High

Target and analytical concentrations reported (Ta-

ble 4). Exposure chamber concentrations were con-

tinuously sampled and the concentration determined

approximately every 40 seconds by FTIR analysis for

each entire 6 hour exposure.

Metric 10:

Exposure Frequency and Duration

High

Exposure duration consistent with cited guideline

(OECD 412)

Metric 11:

Number of Exposure Groups and Dose Spac-

High

Three exposure groups plus concurrent controls were

ing

used (consistent with guideline (OECD 412).. Meth-

ods section states that exposure levels were based
on levels in the accompanying acute (6-hr) study).
However, the discussion states that based on a gen-
eral lack of findings in acute study, the exposure
levels were based on the Kasai et al. (2008) 13-wk
study. Doses selected showed dose-response findings,
and are considered appropriate.

Continued on next page . ..

Page 32 of 187

-------
. continued from previous page

Study Citation: Mattie, DR; Bucher, TW; Carter, AL; Stoffregen, DE; Reboulet, JE (2012). Acute inhalation toxicity study of 1, 4-dioxane in rats

(Rattus norvegicus) GRA and 1(20), 29
Data Type: 2-wk inhalation study - systemic effects

HERO ID: 3563367

Domain

Metric

Rating^ MWF* Score

Comments^

Metric 12: Exposure Route and Method

High

x 1

Domain 4: Test Organism

Metric 13: Test Animal Characteristics

High

Metric 14: Adequacy and Consistency of Animal Hus- High
bandry Conditions

Metric 15: Number per Group High

X 2 2 Albino inbred Fischer (CDF®) [F344/DuCrl] rats.

Age not reported. Based on weights (150-200g for
males, 125-175g for females) they were young adults.

X 1 1 Husbandry conditions were the same between

groups. All animals acclimated to exposure cham-
bers for 5 days before exposure.

X 1 1 16/sex/group; 8/sex sacrificed at end of exposure,

8/sex sacrificed 2 weeks after exposure (minimum
guideline: 5/sex/group per sacrifice)

Domain 5: Outcome Assessment

Metric 16: Outcome Assessment Methodology

High

Metric 17
Metric 18
Metric 19

Consistency of Outcome Assessment
Sampling Adequacy
Blinding of Assessors

Metric 20: Negative Control Response

High
High

Not Rated

High

X 2 2 Hepatic, Renal - Clinical chemistry, OW, HP

Respiratory - HP of entire respiratory tract, includ-
ing nasal sections (Cited guideline indicates that
BALF should be done; however, study authors did
not indicate that this was done. The extensive
histopathological evaluation is considered adequate
to assess this endpoint)

Hematology - at sacrifice

X 1 1 Assessment identical across groups.

X 1 1 Sampling consisted with cited guideline (OECD 412)

NA NA Only non-subjective outcomes and initial

histopathological evaluations performed; blind-
ing not necessary.

X 1 1 Quantitative lesion data reported. Qualitative state-

ment regarding no statistically significant changes
in clinical chemistry or hematology covers both con-
trol and exposure groups. Organ weight data not
reported for any group (downgraded in data presen-
tation metric, not here)

Domain 6: Confounding / Variable Control

Continued on next page

Page 33 of 187

-------
. continued from previous page

Study Citation: Mattie, DR; Bucher, TW; Carter, AL; Stoffregen, DE; Reboulet, JE (2012). Acute inhalation toxicity study of 1, 4-dioxane in rats

(Rattus norvegicus) GRA and 1(20), 29
Data Type:	2-wk inhalation study - systemic effects

HERO ID:	3563367

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 21:

Confounding Variables in Test Design and

Low

x 2

6

Methods section states that evaluations of respi-



Procedures







ration were conducted, but respiratory rate was









not reported (no reporting of clinical signs, or lack
thereof). Rated as low since 1,4-dioxane is a respi-
ratory irritant.

Metric 22:

Health Outcomes Unrelated to Exposure

Medium

x 1

2

No mortalities were reported. Unlike Acute study,
no mention of potential environmental irritants or
infection. Because those confounders were reported
in the acute study (and not specifically addressed in
subacute study), I rated as medium.

Domain 7: Data Presentation and Analysis









Metric 23:

Statistical Methods

High

x 1

1

Lesion incidence compared with Fisher's exact test.









Continuous data analyzed by t-test and ANOVA.

Metric 24:

Reporting of Data

Medium

x 2

4

Quantitative reporting of lesions. Qualitative neg-
ative result reporting for hematology and clinical
chemistry. Incidence data reported, but individual
animal histopathology results not reported. Organ
weights not reported. Likely no effect (no impact on
outcome), so rated as medium.

Overall Quality Determination1"

High

1.3

Extracted

Yes





MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

J]. (Metric Score; x MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 34 of 187

-------
Table 11: Animal toxicity evaluation results of Dow et al 1989 for a repeat dose in vivo DNA synthesis study on hepatic, genotoxicity,
body weight outcomes

Study Citation: Dow Chemical Company (1989). Differentiation of the mechanisms of oncogenicity of 1,4-dioxane and 1,3-hexachlorobutadiene in the
rat

Data Type: Repeat dose in vivo DNA synthesis

HERO ID: 4158030

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

Low

Reported only as "1,4-dioxane".

Metric 2:

Test Substance Source

Medium

Baker Chemical Company; no batch number, but

purity was analyzed by study laboratory

Metric 3:

Test Substance Purity

High

>99%

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

Concurrent vehicle (saline) control was used

Metric 5:

Positive Controls

High

Known genotoxic agent dimethylnitrosamine (DMN)

was used as a positive control

Metric 6:

Randomized Allocation

High

Animals were computer randomized into treatment

groups in all experiments

Domain 3: Exposure Characterization

Metric 7:

Preparation and Storage of Test Substance

Low

Storage details not reported. Mixed with drinking

water. No details on frequency of drinking water

preparation.

Metric 8:

Consistency of Exposure Administration

High

Exposure conditions consistent between groups.

Metric 9:

Reporting of Doses/Concentrations

Low

Study authors report drinking water provided an av-

erage dose of 0, 10, or 1000 mg/kg-d. Nominal doses

in drinking water were not reported. Data used to

calculate average daily dose was not provided.

Metric 10:

Exposure Frequency and Duration

High

11 weeks, 7d/wk

Metric 11:

Number of Exposure Groups and Dose Spac-

High

2 doses plus negative and positive control

Metric 12:

ing

Exposure Route and Method

Medium

No rationale was provided for switching from gavage

(accompanying acute study) to repeat-dose drink-

ing water study. Other compounds (HCBD, DMN)

were administered via gavage for both studies. How-

ever, BWG was decreased by —45-55% following sin-

gle gavage administration of 1000 mg/kg; this BW

effect was not observed with drinking water admin-

istration of 1000 mg/kg over 11 weeks. SO perhaps
the change in route was due to the decreased body
weight associated with gavage administration.

Domain 4: Test Organism

Continued on next page . ..

Page 35 of 187

-------
.. . continued from previous page

Study Citation: Dow Chemical Company (1989). Differentiation of the mechanisms of oncogenicity of 1,4-dioxane and 1,3-hexachlorobutadiene in the
rat

Data Type: Repeat dose in vivo DNA synthesis

HERO ID: 4158030

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 13:

Test Animal Characteristics

High

Male SD rats (Spartan Research). Based on weight
(180-260g), they were adult animals.

Metric 14:

Adequacy and Consistency of Animal Hus-
bandry Conditions

Medium

Husbandry was consistent between groups (wire
cages, environmentally controlled rooms, food and
water ad libitum). Number of rats/cage was not re-
ported, environmental conditions not reported.

Metric 15:

Number per Group

High

5-6/group

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

Genotox, organ weight, and histology of liver (cancer
target organ); body weight and food consumption
also monitored.

Metric 17:

Consistency of Outcome Assessment

High

Consistent evaluation across study groups

Metric 18:

Sampling Adequacy

High

5-6/group

Metric 19:

Blinding of Assessors

Not Rated

Only non-subjective and initial histological evalua-
tions; blinding not required.

Metric 20:

Negative Control Response

High

negative control response was reported; no devia-
tions from normal were reported.

Domain 6: Confounding / Variable Control

Metric 21:

Confounding Variables in Test Design and
Procedures

High

Initial BW 180-260g (not reported per group). Body
weight gains similar between groups during study.

Metric 22:

Health Outcomes Unrelated to Exposure

Medium

data on attrition and/or health outcomes unrelated
to exposure for each study group were not reported
because only substantial differences among groups
were noted

Domain 7: Data Presentation and Analysis

Metric 23:

Statistical Methods

Medium

Continuous data were compared by Dunnett's t-test.
No statistical analysis of histopathological findings.
Histological findings only reported qualitatively.

Metric 24:

Reporting of Data

High

DNA synthesis, liver weight, and BWG reported
quantitatively with statistics. Histopathological

findings reported qualitatively (present or absent at
dose).

Overall Quality Determination1"

High —~ Medium5

Extracted

Yes

Continued on next page . ..

Page 36 of 187

-------
. continued from previous page

Study Citation: Dow Chemical Company (1989). Differentiation of the mechanisms of oncogenicity of 1,4-dioxane and 1,3-hexachlorobutadiene in the
rat

Data Type:	Repeat dose in vivo DNA synthesis

HERO ID:	4158030

Domain	Metric	Rating^ MWF* Score	Comments^

* MWF = Metric Weighting Factor

t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

+ The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

if any metric is Unacceptable

Overall rating =

J]. (Metric Score; x MWF;) / J] . MWFj

(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

§ Evaluator's explanation for rating change: "Downgraded based on the uncertainty in the actual doses (see metric 9)."

Page 37 of 187

-------
Table 12: Animal toxicity evaluation results of Itoh 2019 - in vivo genotoxicity assay - micronucleus test

Study Citation: Itoh, S; Hattori, C (2019). In vivo genotoxicity of 1,4-dioxane evaluated by liver and bone marrow micronucleus tests and Pig-a assay

in rats Mutation Research: Genetic Toxicology and Environmental Mutagenesis, 837 8-14
Data Type:	In vivo genotox assays

HERO ID:	5072318

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance













Metric 1:

Test Substance Identity

High

X

2

2

1,4-dioxane (CAS No. 123-91-1)

Metric 2:

Test Substance Source

High

X

1

1

Wako Pure Chemical Industries, Ltd. (Osaka,











Japan)

Metric 3:

Test Substance Purity

Low

X

1

3

The purity and/or grade were not reported

Domain 2: Test Design













Metric 4:

Negative and Vehicle Controls

High

X

2

2

Concurrent vehicle control

Metric 5:

Positive Controls

High

X

1

1

For liver micronucleus: diethylnitrosamine [DNN]
(juvenile and partial hepatectomy methods), car-
bendazim (partial hepatectomy method)

Bone marrow micronucleus: cyclophosphamide
monohydrate [CP]

Pig-a assay: 7,12-dimethylbenz[a]anthracene

[DMBA]

Metric 6:

Randomized Allocation

Low

X

1

3

The study did not report how animals were allocated
to study groups

Domain 3: Exposure Characterization











Metric 7:

Preparation and Storage of Test Substance

High

X

1

1

The test chemical and DEN were dissolved in water
for injection. Carbendazin was suspended on 0.5%
methylcellulose. CP and DMBA were dissolved and
suspended in saline.

Metric 8:

Consistency of Exposure Administration

High

X

1

1

Exposure administration was consistent across ex-
posure groups for each experiment.

Metric 9:

Reporting of Doses/Concentrations

High

X

2

2

0, 1000, 2000, or 3000 mg/kg

Metric 10:

Exposure Frequency and Duration

High

X

1

1

Liver MN juvenile method: dosed on Day 1 and Day
2, hepatocyte isolation on Day 6

Liver-MN PH method: Exposed once either the day
before PH or day after PH; hepatocyte isolation 5
days after PH

Metric 11: Number of Exposure Groups and Dose Spac- High	x 1	1

ing

Metric 12: Exposure Route and Method	High	x 1	1

Bone marrow MN: Exposed once (Day 1) with bone
marrow removed Day 2 or 3

Pig-a test: Exposed once (Day 1) with peripheral
blood obtained on Days -1, 15, and 30

0, 1000, 2000, or 3000 mg/kg based on previous re-
ports

Gavage at dose volume of 10 mL/kg

Continued on next page

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.. . continued from previous page

Study Citation: Itoh, S; Hattori, C (2019). In vivo genotoxicity of 1,4-dioxane evaluated by liver and bone marrow micronucleus tests and Pig-a assay

in rats Mutation Research: Genetic Toxicology and Environmental Mutagenesis, 837 8-14
Data Type:	In vivo genotox assays

HERO ID:	5072318

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 4: Test Organism











Metric 13:

Test Animal Characteristics

High

x 2

2

Male F344/DuCrlCrlj rats, 4- to 8-wks of age;











Charles River Laboratories Japan, Inc.

Metric 14:

Adequacy and Consistency of Animal Hus-

High

x 1

1

This study was conducted in compliance with the



bandry Conditions







following law and









guidelines; "Law Concerning the Protection and











Control of Animals",











Japanese Law No. 105, October 1, 1973, revised on











June 22, 2005

Metric 15:

Number per Group

High

x 1

1

4-5/group per test

Domain 5: Outcome Assessment









Metric 16:

Outcome Assessment Methodology

High

x 2

2

High for genotoxicity: evaluated with 4 tests - two











liver MN assays, a bone marrow MN assay, and











blood Pig-a mutation assay











Unacceptable for liver toxicity (only relative











liver weight evaluated)

Metric 17:

Consistency of Outcome Assessment

High

x 1

1



Metric 18:

Sampling Adequacy

High

x 1

1



Metric 19:

Blinding of Assessors

Not Rated

NA

NA

All quantitative measures

Metric 20:

Negative Control Response

High

x 1

1



Domain 6: Confounding / Variable Control









Metric 21:

Confounding Variables in Test Design and

Medium

x 2

4

Initial BW not reported; not likely to have substan-



Procedures







tial impact

Metric 22:

Health Outcomes Unrelated to Exposure

High

x 1

1



Domain 7: Data Presentation and Analysis









Metric 23:

Statistical Methods

High

x 1

1

MN: two-tailed Fisher's exact test and two-tailed









Cochran-Armitage trend test











% IE: Wilcoxon's rank sum











Pig-a: Bartlett's test to evaluate the homogeneity of











variance; analyzed by a parametric Dunnett's test











when the variance was homogeneous or by a Steel's











(nonparametric Dunnett's) test when it was not

Metric 24:

Reporting of Data

High

x 2

2

Graphical reporting of all genotox data; quantitative











reporting for relative liver weight data

Overall Quality Determination



High



1.2



Extracted



Yes







Continued on next page . ..

Page 39 of 187

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. continued from previous page

Study Citation: Itoh, S; Hattori, C (2019). In vivo genotoxicity of 1,4-dioxane evaluated by liver and bone marrow micronucleus tests and Pig-a assay

in rats Mutation Research: Genetic Toxicology and Environmental Mutagenesis, 837 8-14
Data Type:	In vivo genotox assays

HERO ID:	5072318

Domain	Metric	Rating^ MWF* Score	Comments^

* MWF = Metric Weighting Factor

t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

+ The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

if any metric is Unacceptable

Overall rating =

J]. (Metric Score; x MWF;) / J] . MWFj

(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 40 of 187

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3 Subchronic (30-90 days)

Table 13: Animal toxicity evaluation results of Kasai et al 2008 for a 13-week inhalation study on hepatic, renal, hematology, clinical
chemistry, respiratory, body weight, mortality outcomes

Study Citation: Kasai, T; Saito, M; Senoh, H; Umeda, Y; Aiso, S; Ohbayashi, H; Nishizawa, T; Nagano, K; Fukushima, S (2008). Thirteen-week

inhalation toxicity of 1,4-dioxane in rats Inhalation Toxicology, 20(10), 961-971
Data Type:	13-week inhalation

HERO ID:	195044

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance











Metric 1:

Test Substance Identity

High

x 2

2

Reagent grade 1,4-Dioxane (>99% pure); liquid

Metric 2:

Test Substance Source

High

x 1

1

Obtained from Wako Pure Chemical Industries, Ltd.









(Osaka, Japan). Batch number not provided, but
identity and composition verified by laboratory us-
ing GC-MS.

Metric 3:

Test Substance Purity

High

x 1

1

Reagent grade 1,4-Dioxane (>99% pure); analyzed
for purity and stability using GC-MS before and af-
ter use. Butylhydoxytoluene was detected in 1,4-
dioxane liquid by GC-MS (1.3 ppm w/w), but it was
not detected in air samples collected from inhalation
air samples.

Domain 2: Test Design











Metric 4:

Negative and Vehicle Controls

High

x 2

2

Concurrent control group exposed to clean air under
same conditions as test groups.

Metric 5:

Positive Controls

Not Rated

NA

NA

Positive control group is not needed in standard 13-
wk inhalation study (see OECD guideline 413)

Metric 6:

Randomized Allocation

High

x 1

1

stratified randomization into 8 body-weight-









matched groups, each comprised of 10 rats/sex

Domain 3: Exposure Characterization









Metric 7:

Preparation and Storage of Test Substance

High

x 1

1

Detailed description of vapor generation; chamber
concentrations of 1,4-dioxane monitored every 15
minutes during exposure;

Metric 8:

Consistency of Exposure Administration

High

X 1

1

Exposure conditions identical between groups (ex-
cept exposure levels). All animals in an exposure
group were exposed simultaneously (exposure cham-
ber held 20 individual cages).



Continued on

next page . .







Page 41 of 187

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.. . continued from previous page

Study Citation: Kasai, T; Saito, M; Senoh, H; Umeda, Y; Aiso, S; Ohbayashi, H; Nishizawa, T; Nagano, K; Fukushima, S (2008). Thirteen-week

inhalation toxicity of 1,4-dioxane in rats Inhalation Toxicology, 20(10), 961-971
Data Type: 13-week inhalation

HERO ID: 195044

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 9:

Reporting of Doses/Concentrations

High

Analytical concentrations reported, and within 1%
of target. Chamber concentrations of 1,4-dioxane
monitored every 15 minutes during exposure. Ac-
curacy and precision of the actual concentrations
of 1,4-dioxane in the exposure chamber were kept
by periodic injection of the certified standard 1,4-
dioxane gas (Takachiho Co., Ltd., Tokyo) into the
gas chromatograph for the calibration curve of 1,4-
dioxane.

Metric 10:

Exposure Frequency and Duration

High

Consisted with cited OECD guideline 413 (6 h/d, 5
d/wk, 13 wk)

Metric 11:

Number of Exposure Groups and Dose Spac-

Medium

Adequate number of exposure groups (n=7 plus con-

ing

trol). However, lowest dose was identified as a

LOAEL (no NOAEL identified), and the highest
dose was 100% lethal (high dose too high). How-
ever, the number of dose groups provides dose re-
sponse data (increased effects/incidence with in-
creasing dose).

Metric 12:

Exposure Route and Method

High

Detailed description of vapor generation and whole-
body exposure conditions (1060 L exposure cham-
bers, housed 20 individual cages).

Domain 4: Test Organism

Metric 13:

Test Animal Characteristics

High

Six-week-old F344/DuCrj rats of both sexes (ob-

tained at 4-weeks of age)

Metric 14:

Adequacy and Consistency of Animal Hus-
bandry Conditions

High

Housing conditions described adequately; same con-
ditions in control and exposure groups.

Metric 15:

Number per Group

High

10/sex/group, as per cited OECD guideline 413

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

PECO endpoints:

Renal - clinical chemistry, urinalysis, organ weight,
histology

Hepatic - clinical chemistry, urinalysis, organ
weight, histology

Neuro - clinical signs, brain, spinal cord, and nerve

histo, assumed brain weight due to cited OECD 413

guideline

Other endpoints:

Respiratory - lung weight, histo of entire respiratory

tract (including nasal sections)

Hemato, BW, mortality - adequately evaluated

Continued on next page . ..

Page 42 of 187

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.. . continued from previous page

Study Citation: Kasai, T; Saito, M; Senoh, H; Umeda, Y; Aiso, S; Ohbayashi, H; Nishizawa, T; Nagano, K; Fukushima, S (2008). Thirteen-week

inhalation toxicity of 1,4-dioxane in rats Inhalation Toxicology, 20(10), 961-971
Data Type: 13-week inhalation

HERO ID: 195044

Domain Metric Rating^ MWF* Score Comments^

Metric 17:

Consistency of Outcome Assessment

High

Outcomes were assessed consistently across study
groups as described in methods section with excep-
tion of high-dose group due to 100% lethality by
week 1 (histology was performed at death). There
were no mortalities in other groups. Due to 6 ex-
posure groups other than the high-dose group, loss
of this high dose group to 13 week assessments does
not alter evaluation or interpretation of the results.

Metric 18:

Sampling Adequacy

High

Sampling consistent with cited OECD guideline 413.

Metric 19:

Blinding of Assessors

Not Rated

Blinding status of assessors was not reported, Evalu-

ated endpoints included non-subjective metrics and

initial histopathology review, so blinding was not

needed.

Metric 20:

Negative Control Response

High

Control results were reported, and within expected
biological variation.

Domain 6: Confounding / Variable Control

Metric 21:

Confounding Variables in Test Design and

Medium

Initial groups were weight-matched. No abnormal

Procedures

clinical signs were reported in surviving groups (all

high-dose animals died within a week), so altered
breathing with exposure is unlikely. However, respi-
ratory rate (or lack of bradypnea) was not specifi-
cally mentioned so I downgraded to medium.

Metric 22:

Health Outcomes Unrelated to Exposure

High

Mortality was limited to the high-exposure group,
and was attributed to exposure-related effects (renal
failure)

Domain 7: Data Presentation and Analysis

Metric 23:

Statistical Methods

High

Continuous variables were evaluated using Dun-

nett's test and dichotomous variables were evaluated
using chi-square. 2-sided analysis with p-values of
0.05 and 0.01 was performed.

Continued on next page . ..

Page 43 of 187

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. continued from previous page

Study Citation: Kasai, T; Saito, M; Senoh, H; Umeda, Y; Aiso, S; Ohbayashi, H; Nishizawa, T; Nagano, K; Fukushima, S (2008). Thirteen-week

inhalation toxicity of 1,4-dioxane in rats Inhalation Toxicology, 20(10), 961-971
Data Type:	13-week inhalation

HERO ID:	195044

Domain

Metric

Rating^ MWF* Score

Comments^

Metric 24: Reporting of Data

Medium

x 2

Only some of the blood parameters (clinical chem-
istry, hematology) were reported quantitatively. It is
assumed that other parameters listed in OECD 413
were evaluated and no exposure-related effects were
found, but results were not reported. A slight de-
crease in urinary protein was qualitatively reported;
no other urinalysis results were reported (again, as-
sumed that endpoints in OECD 413 were evaluated).
Relative organ weights and histology were reported
quantitatively (for exposure-related effects). Male
kidney and male and female nervous system histol-
ogy were not reported, but it is implied that no
exposure-related effects were observed other than
respiratory tract and liver in males and females and
kidneys in females (see histopathology section in re-
sults).

Overall Quality Determination"1'

High

1.2

Extracted

Yes





MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; X MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 44 of 187

-------
Table 14: Animal toxicity evaluation results of Kano et al 2008 for a 13-week oral toxicity of 1,4-d in rats and mice study

Thirteen-week oral

Study Citation: Kano, H; Umeda, Y; Saito, M; Senoh, H; Ohbayashi, H; Aiso, S; Yamazaki, K; Nagano, K; Fukushima, S (2008).

toxicity of 1,4-dioxane in rats and mice Journal of Toxicological Sciences, 33(2), 141-153
Data Type: 13-week oral toxicity of 1,4-D in rats and mice

HERO ID: 196245

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance
Metric 1:

Metric 2:

Metric 3:

Test Substance Identity
Test Substance Source
Test Substance Purity

High
High
High

x 2
x 1
x 1

2 Test substance identified by name; no concern with

different forms or mixtures.

1 Test substance obtained from commercial source.

and its purity established by IS and GC.

1 Test substance obtained from commercial source;

purity >99.0% verified by IS and GC.

Domain 2: Test Design

Metric 4:

Metric 5:
Metric 6:

Negative and Vehicle Controls

Positive Controls
Randomized Allocation

High

Not Rated
High

x 2

NA
x 1

2 Control group received vehicle (deionized water); all

groups were body-weight matched (stratified ran-
domization).

NA Not indicated for study type.

1 Group assignments by stratified randomization into

body-weight matched groups.

Domain 3: Exposure Characterization

Metric 7: Preparation and Storage of Test Substance

High

x 1

Metric 8: Consistency of Exposure Administration

Metric 9: Reporting of Doses/Concentrations

Metric 10: Exposure Frequency and Duration

High

Metric 11: Number of Exposure Groups and Dose Spac- High
ing

x 1
x 2

x 1
x 1

Test material was analyzed for stability before and
after use; no decomposition products or impurities
identified. Test material prepared twice per week.
Analysis of test material immediately after prepa-
ration showed concentrations 94.6-102.9% of target;
analysis of test material 4 days after preparation
showed concentrations 92.8-101.1% of initial concen-
trations.

Daily water intake calculated as difference between
weight of water remaining in bottle 3-4 days after
preparation divided by number of days.

Intake of 1,4-D was estimated by study authors
based on nominal concentration, body weight (mea-
sured once weekly), and water intake (measured ev-
ery 3-4 days).

Frequency was not specified but is inferred to be 7
days per week; duration specified as 13 weeks.

The rationale for dose selection was not stated, but
the study included 5 non-zero exposure concentra-
tions across a 39-fold range. Exposure levels in-
cluded those high enough to induce effects and low
enough to identify a NOAEL.

Continued on next page . ..

Page 45 of 187

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. continued from previous page

Study Citation: Kano, H; Umeda, Y; Saito, M; Senoh, H; Ohbayashi, H; Aiso, S; Yamazaki, K; Nagano, K; Fukushima, S (2008). Thirteen-week oral

toxicity of 1,4-dioxane in rats and mice Journal of Toxicological Sciences, 33(2), 141-153
Data Type: 13-week oral toxicity of 1,4-D in rats and mice

HERO ID: 196245

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 12:

Exposure Route and Method

High

Exposure route was reported and appropriate
(drinking water).

Domain 4: Test Organism

Metric 13:

Test Animal Characteristics

High

Test animal species, strain, age, and source were

all reported and appropriate for subchronic toxicity
evaluation.

Metric 14:

Adequacy and Consistency of Animal Hus-
bandry Conditions

High

No differences between groups in animal husbandry
conditions were reported. Animals were housed in-
dividually.

Metric 15:

Number per Group

High

Study used 10 animals/sex/group, which exceeds
numbers recommended by OECD (5/sex/grp)

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

Outcome assessment was described in detail in-

cluding organs/endpoints, methods, instrumenta-
tion, stains, and timing. Endpoints evaluated were
sensitive for systemic toxicity.

Metric 17:

Consistency of Outcome Assessment

High

No inconsistencies in protocol execution were noted

in the report.

Metric 18:

Sampling Adequacy

High

All standard endpoints were evaluated in all animals
of all exposure groups. ALtered hepatic foci evalu-
ated in subsets of high exposure and control groups.

Metric 19:

Blinding of Assessors

Not Rated

There were no subjective outcomes evaluated.

Metric 20:

Negative Control Response

High

Adequately reported.

Domain 6: Confounding / Variable Control

Metric 21: Confounding Variables in Test Design and Unacceptable x 2
Procedures

Metric 22: Health Outcomes Unrelated to Exposure

High

x 1

In both male and female rats and mice, drinking
water intakes in the top two exposure groups were
at least 20% lower than control intakes.

Animal attrition was limited to two deaths (one rat
and one mouse). No infections or other health out-
comes unrelated to exposure were reported.

Domain 7: Data Presentation and Analysis

Metric 23: Statistical Methods

Metric 24: Reporting of Data

High
High

x 1
x 2

1 Statistical methods were clearly described and ap-
propriate for the data.

2 Data for all groups on exposure-related findings were
reported. Measures of variation and numbers of an-
imals examined were reported.

Overall Quality Determination1"

Unacceptable*

Medium5 ir3-

Continued on next page . ..

Page 46 of 187

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. continued from previous page

Study Citation: Kano, H; Umeda, Y; Saito, M; Senoh, H; Ohbayashi, H; Aiso, S; Yamazaki, K; Nagano, K; Fukushima, S (2008). Thirteen-week oral

toxicity of 1,4-dioxane in rats and mice Journal of Toxicological Sciences, 33(2), 141-153
Data Type:	13-week oral toxicity of 1,4-D in rats and mice

HERO ID:	196245

Domain

Metric

Rating^

MWF*

Score

Comments^

Extracted

Yes

* Consistent with our Application of Systematic Review in TSCARisk Evaluations document, if a metric for a data source receives a score of Unacceptable (score = 4), EPA will
determine the study to be unacceptable. In this case, one or more of the metrics were rated as unacceptable. As such, the study is considered unacceptable and the score is
presented solely to increase transparency.

MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; X MWF;) /]T . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out
and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

§ Evaluator's explanation for rating change: "Although there was a dose-related decrease in water intake that exceeded 20

Page 47 of 187

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4 Chronic (>90 days)

Table 15: Animal toxicity evaluation results of Argus et al 1965 for a cancer bioassay-liver, kidney, blood study on cancer outcomes

Study Citation: Argus, MF; Arcos, JC; Hoch-Ligeti, C (1965). Studies on the carcinogenic activity of protein-denaturing agents: Hepatocarcinogenicity

of dioxane Journal of the National Cancer Institute, 35(6), 949-958
Data Type:	Cancer bioassay-liver, kidney, blood

HERO ID:	17009

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance
Metric 1:

Metric 2:
Metric 3:

Test Substance Identity

Test Substance Source
Test Substance Purity

High

Medium
Low

x 2

x 1
x 1

2

2

3

Test substance identified by name and chemical for-
mula and structure

Eastman organic chemical number was reported
Purity was not reported

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

Low

x 2

6

Details regarding the negative control group were
not reported, based on the study design, it is not
clear that the animals were treated in any manner
making direct comparison among results challeng-
ing.

The metric is not applicable.

How animals were allocated was not reported.

Metric 5:
Metric 6:

Positive Controls
Randomized Allocation

Not Rated
Low

NA
x 1

NA
3

Domain 3: Exposure Characterization

Metric 7: Preparation and Storage of Test Substance

Medium

x 1

2

Limited preparation (1% in drinking water) informa-
tion was reported and storage information was not
provided. Given that 1,4-dioxane is stable in water,
the incomplete information is not expected to have
a substantial impact on results.

Metric 8: Consistency of Exposure Administration	High	x 1

Metric 9: Reporting of Doses/Concentrations	Medium x 2

Metric 10: Exposure Frequency and Duration	High	x 1

Metric 11: Number of Exposure Groups and Dose Spac-	Not Rated	NA
ing

Metric 12: Exposure Route and Method	High	x 1

1	Treated animals had access to drinking water con-

tinuously

4 The maximum dose/rat, approximate daily water in-
take rate, and body weight range at the end of the
study were reported, so approximation of dose could
be calculated.

1	Data found in Table 1.

NA Only one treatment dose was used

Exposure through drinking water was acceptable as
1,2-dioxane can leach into and remain in water

Domain 4: Test Organism









Metric 13:

Test Animal Characteristics Medium

x 2

4

Animal source, species, strain, sex, life-stage, and









body weight range were reported. Specific age and









health status was not reported.

Continued on next page . ..

Page 48 of 187

-------
. continued from previous page

Study Citation: Argus, MF; Arcos, JC; Hoch-Ligeti, C (1965). Studies on the carcinogenic activity of protein-denaturing agents: Hepatocarcinogenicity

of dioxane Journal of the National Cancer Institute, 35(6), 949-958
Data Type: Cancer bioassay-liver, kidney, blood

HERO ID: 17009

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 14:

Adequacy and Consistency of Animal Hus-
bandry Conditions

Medium

Limited husbandry conditions were reported, but
appear to be similar among the groups.

Metric 15:

Number per Group

Medium

The reported number was lower than the typical
number (26 vs 30 for cancer bioassay). It is unclear
if this is the initial number of animals/group.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

Medium

Limited details regarding the complete necropsy and
histological investigation were reported.

Metric 17:

Consistency of Outcome Assessment

High

Based on the study report, it is inferred that out-
come assessment was consistent.

Metric 18:

Sampling Adequacy

High

Sampling was adequate.

Metric 19:

Blinding of Assessors

Not Rated

This metric is not applicable.

Metric 20:

Negative Control Response

High

Biological responses were adequate.

Domain 6: Confounding / Variable Control

Metric 21:

Confounding Variables in Test Design and
Procedures

Medium

The lack of reported of initial body weight and spe-
cific water intake is not likely to have a substantial
impact on results.

Metric 22:

Health Outcomes Unrelated to Exposure

Low

Data on attrition and/or health outcomes unrelated
to exposure were not reported.

Domain 7: Data Presentation and Analysis

Metric 23:

Statistical Methods

Low

Statistical analysis was not conducted, but some
data were provided which could be used to do an
independent analysis (incidence of rats with tumors)

Metric 24:

Reporting of Data

Medium

Tabular data for tumor outcomes was reported, all

other data were described in the text and incidence
and severity data were not reported.

Overall Quality Determination

Medium

1.9

Extracted

Yes

MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; x MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating,
ft This metric met the criteria for high confidence as expected for this type of study

Page 49 of 187

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Table 16: Animal toxicity evaluation results of Kociba et al 1974 for a 2-year drinking water study study on cancer, hepatic, renal,
hematological and immune, body weight, mortality outcomes

I. Results of a 2-year ingestion study in rats

Study Citation: Kociba, RJ; Mccollister, SB; Park, C; Torkelson, TR; Gehring, PJ (1974). 1,4-dioxane.

Toxicology and Applied Pharmacology, 30(2), 275-286
Data Type: 2-year drinking water study

HERO ID: 62929

Domain

Metric

Rating^ MWF* Score

Comments^

Domain 1: Test Substance
Metric 1:
Metric 2:

Test Substance Identity
Test Substance Source

Metric 3: Test Substance Purity

Higll X 2 2 Clearly identifies substance as 1,4-dioxane

Medium X 1 2 Compound obtained from The Dow Chemical Co.

(batch no. not reported).

High X 1 1 Purity not reported, but stock samples were an-

alyzed for impurities at 6 different times during
2-year study. The following impurities were re-
ported in stock solutions: hydrogen peroxide (10-340
ppm), crotonaldehyde (220-1340 ppm), 2-methyl-
1,3-dioxolane (6-108 ppm), water (10-90 ppm). No
acetaldehyde was detected. So purity was >99%.

Domain 2: Test Design

Metric 4:

Metric 5:
Metric 6:

Negative and Vehicle Controls

Positive Controls
Randomized Allocation

High X 2 2 Untreated controls were given regular drinking wa-

ter.

Not Rated NA NA Positive control not warranted by study type.

Low X 1 3 The study did not report how animals were allocated

to study groups

Domain 3: Exposure Characterization

Metric 7: Preparation and Storage of Test Substance High

Metric 8: Consistency of Exposure Administration High

Metric 9: Reporting of Doses/Concentrations High

Metric 10: Exposure Frequency and Duration High

X 1 1 Storage conditions prior to opening were provided.

Samples were used within 1 week after bottles were
opened. Drinking water solutions were prepared
twice weekly during the first year and weekly during
the second year.

X 1 1 Drinking water was available ad libitum to all expo-

sure groups.

X 2 2 Daily water consumption was recorded, with rates

calculated for 3 different time periods of the 2-year
study (Days 1-113, 114-198, 446-460). These values
plus BW data were used to calculate daily doses of
1,4-dioxane in mg/kg/day. Drinking water samples
were analyzed for 1,4-dioxane content "periodically"
via gas liquid chromatography.

X 1 1 2 yr study; drinking water available ad libitum

Continued on next page ..

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.. . continued from previous page

Study Citation: Kociba, RJ; Mccollister, SB; Park, C; Torkelson, TR; Gehring, PJ (1974). 1,4-dioxane. I. Results of a 2-year ingestion study in rats

Toxicology and Applied Pharmacology, 30(2), 275-286
Data Type: 2-year drinking water study

HERO ID: 62929

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 11:

Number of Exposure Groups and Dose Spac-

High

3 dose groups - low dose did not induce toxic ef-

ing

fects or tumors; mid-dose induced some toxic effects,

high-dose induced tumors.

Metric 12:

Exposure Route and Method

High

drinking water administration

Domain 4: Test Organism

Metric 13:

Test Animal Characteristics

High

6-8 wk old Sherman rats; male and female

Metric 14:

Adequacy and Consistency of Animal Hus-

Medium

Information on husbandry limited to "maintained in

bandry Conditions

animal care facilities fully accredited by the Ameri-

can Association for Accreditation of laboratory An-
imal Care". All rats were maintained under these
"approved conditions". Water and standard feed
available ad libitum.

Metric 15:

Number per Group

High

60/sex/group

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

Cancer: complete histological analysis, sufficient du-
ration of study
Renal: OW, histopathology
Hepatic: OW, histopathology

Hematology, Bd wt, mortality - adequately assessed

Metric 17:

Consistency of Outcome Assessment

High

The same protocols were used for control and expo-
sure groups.

Metric 18:

Sampling Adequacy

High

Adequate numbers were used in all groups. Effective
number of animals for tumor analysis was calculated.

Metric 19:

Blinding of Assessors

Not Rated

All evaluations were non-subjective or initial

histopathological evaluations.

Metric 20:

Negative Control Response

High

Control results reported, no noted deviations from
expectation.

Domain 6: Confounding / Variable Control

Metric 21:

Confounding Variables in Test Design and

High

Based on graphically reported data, BW were sim-

Procedures

ilar between groups at study initiation. Decreased

water consumption was observed in high-dose group
(10-12% during Days 1-198) and mid-dose group fe-
males (8% from days 114-198).

Metric 22:

Health Outcomes Unrelated to Exposure

High

Decreased survival during the first 4 months of the

study in the high-dose group attributed to exposure
(hepatic and renal toxicity); mortality was compa-
rable to control in low- and mid-dose group.

Domain 7: Data Presentation and Analysis

Continued on next page . ..

Page 51 of 187

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. continued from previous page

Study Citation: Kociba, RJ; Mccollister, SB; Park, C; Torkelson, TR; Gehring, PJ (1974). 1,4-dioxane. I. Results of a 2-year ingestion study in rats

Toxicology and Applied Pharmacology, 30(2), 275-286
Data Type:	2-year drinking water study

HERO ID:	62929

Domain

Metric

Rating^ MWF* Score

Comments^

Metric 23: Statistical Methods

Metric 24: Reporting of Data

High

Medium

x 1

x 2

Tumors evaluated using Fisher's Exact probability
test. Survival rates were compared using Chi-Square
and Fisher's Exact probability test. Student t test
was used to compared continuous variables.

Cancer - tumor incidence data reported adequately
Hepatic - significant change in liver weight reported
qualitatively only, nonneoplastic changes reported
qualitatively only

Renal - no change in OW (qualitative), nonneoplas-
tic changes reported qualitatively only
Hematological - no change in parameters (qualita-
tive)

Bd wt and Mortality reported graphically

Overall Quality Determination1"

High

1.2

Extracted

Yes





MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; X MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 52 of 187

-------
Table 17: Animal toxicity evaluation results of NCI et al 1978 for a cancer bioassay- male rats study on cancer outcomes

Study Citation: NCI (1978).

Bioassay of 1,4-dioxane for possible carcinogenicity

Data Type: Cancer bioassay- male rats

HERO ID: 62935

Domain

Metric

Rating^

MWF*

Score

('o m mo ill &TT

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

x 2

The test substance was identified by name and

CASRN.

Metric 2:

Test Substance Source

High

x 1

The source of the test substance was reported, in-

cluding lot numbers. The test substance (one of two
lots) was analyzed to confirm identity and purity
(using vapor phase chromatography and spectrome-

try).

Metric 3:

Test Substance Purity

High

x 1

The purity (one of two lots) was 99.9%. The test
substance was tested for specific impurities (sodium
diethylthiocarbamate, and peroxide); these impuri-
ties were generally present at 0.001% or less. How-
ever one lot showed peroxide levels of 0.1% after
study completion. This deficiency is not likely to
substantially impact the study results.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

Unacceptable

x 2

Matched drinking water control groups were used.
However, groups were not placed on study at the
same time. Control and high-dose male rats were
placed on study later than other groups (by 1 year).
Based on data presented graphically in the study re-
port, the weights of low-dose male rats differed from
the body weights of control and high-dose animals
at study week 0.

Metric 5:

Positive Controls

Not Rated

Positive control group not indicated by study type.

Metric 6:

Randomized Allocation

Low

x 1

Animals were assigned to control or dose groups "ac-
cording to a series of random numbers;" there were
deficiencies regarding the allocation method that
may impact the study results (e.g. allocation by
animal number).

Domain 3: Exposure Characterization

Metric 7:

Preparation and Storage of Test Substance

Medium

x 1

Test substance preparation and storage conditions
were not reported in exhaustive detail ("dioxane so-
lutions prepared in tap water twice per week and
stored in polyethylene containers"). Test substance
stability was demonstrated via analyses conducted
several months after study completion; however,
data on stability of the test substance under the
conditions of administration (in water) were not pro-
vided.

Continued on

next page . ..

Page 53 of 187

-------
. continued from previous page

Study Citation: NCI (1978). Bioassay of 1,4-dioxane for possible carcinogenicity
Data Type:	Cancer bioassay- male rats

HERO ID:	62935

Domain

Metric

Rating^

MWF*

Score

('o m mo ill &TT

Metric 8:

Consistency of Exposure Administration

High

X

1

1

Dosed water or tap water was available ad libitum.

Metric 9:

Reporting of Doses/Concentrations

Low

X

2

6

As per applicable guideline, water consumption
should be measured at least weekly for the first 13
weeks and at least monthly thereafter. Although
doses in mg/kg-day were provided, these doses were
based on water consumption determined at intervals
during the second year of the bioassay only (and
using 20% of the animals as a representative sam-
ple). The study report indicates that "there were
wide fluctuations in intake at different time periods
within groups."

Metric 10:

Exposure Frequency and Duration

High

X

1

1

As per applicable guideline, the duration of the
study will normally be 24 months for rats . In this
study, rats were dosed for 110 weeks.

Metric 11:

Number of Exposure Groups and Dose Spac-

Low

X

1

3

Concentrations were chosen based on the results of



ing









previous studies (by Argus et al. 1965). As per











applicable guideline, at least three dose levels and
a concurrent control should be used; however, the
PECO statement requires at least 2 dose groups and
a control. The study used two dose groups and a
control; however, the control groups was not con-
current (i.e.. data for only 1 quantitative dose group
and controls in male rats were concurrent). The dif-
ference between the low- and high-dose in rats was
also not two-fold (as intended). These factors are
likely to have an impact on the study results.

Metric 12:

Exposure Route and Method

Medium

X

1

2

The route of exposure was reported (i.e. drinking
water); however, no rationale was provided. The
applicable guideline considers drinking water to be
a valid route of administration.

Domain 4: Test Organism













Metric 13:

Test Animal Characteristics

High

X

2

2

The test animal species, strain, health status, sex,











age, and body weights at study week 0 (provided
graphically) were reported. Animals were obtained
from a commercial laboratory. These animals were
appropriate models for the evaluation of carcino-
genicity (although not the same rat strain used in
previous studies).

Continued on next page . ..

Page 54 of 187

-------
.. . continued from previous page

Study Citation: NCI (1978). Bioassay of 1,4-dioxane for possible carcinogenicity
Data Type:	Cancer bioassay- male rats

HERO ID:	62935

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 14:

Adequacy and Consistency of Animal Hus-

High

x 1

1

Husbandry conditions (temperature, humidity, light



bandry Conditions







cycles) were reported, and appear to be adequate









(compared to guideline recommendations;) and the
same for control and dosed groups. The applicable
guideline indicates that animals should be housed
individually or in small groups. The study report
indicates that rats were housed 4 per cage. This is
unlikely to have had a substantial impact on results
(there were no indications of injuries or death due
to overcrowding).

Metric 15:

Number per Group

Medium

X 1

2

The number of animals per study group was
lower than the typical number used in carcino-
genicity studies in rats (35/sex/group compared to
50/sex/group recommended by guideline). However,
the study report indicated that animal numbers were
adequate for statistical analyses (related to carcino-
genicity).

Domain 5: Outcome Assessment









Metric 16:

Outcome Assessment Methodology

Medium

x 2

4

Animals from all dose groups were subjected to gross
and microscopic pathology evaluations. The num-
ber of tissues evaluated was not as comprehensive as
that recommended by guideline (at least in low-dose
rats), but this deficiency is not likely to substantially
impact the study results.

Metric 17:

Consistency of Outcome Assessment

Medium

x 1

2

Surviving rats were sacrificed at 110-117 weeks. The









tissues from some animals were not evaluated (par-
ticularly in animals that died early). Therefore, the
numbers of animals subjected to histopathological
evaluations (with respect to specific organs or tis-
sues) are not the same as the number of animals
placed on study.

Metric 18:

Sampling Adequacy

Medium

x 1

2

Histopathological examinations were performed on
dosed groups and controls. Although details were
not reported (e.g. the numbers of slides evaluated,
individual animal data available but not provided),
these deficiencies are not likely to substantially im-
pact the study results.

Metric 19:

Blinding of Assessors

Not Rated

NA

NA

Blinding not reported, but is not required for initial
histopathology review.

Metric 20:

Negative Control Response

High

x 1

1

The biological responses of the negative control

groups were adequate (showing no or low incidences
of lesions).

Domain 6: Confounding / Variable Control

Continued on next page . ..

Page 55 of 187

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. continued from previous page

Study Citation: NCI (1978). Bioassay of 1,4-dioxane for possible carcinogenicity
Data Type:	Cancer bioassay- male rats

HERO ID:	62935

Domain

Metric

Rating^

MWF*

Score

('o m me ill &TT

Metric 21:

Confounding Variables in Test Design and
Procedures

Low

x 2

6

Doses administered to low- and high-dose groups of
rats were not reflective of the intended doses ow-
ing (at least in part) to decreased palatability (wa-
ter consumption data were not provided). Initial
body weights were not explicitly reported (weights
at study week 0 were shown graphically). Rats
were housed in the same room with rats admin-
istered dibenzodioxin, 2,7-dichlorobenzodioxin, and
1,2,3,4,6,7,8,9-octachlorodibenzodioxin.

Metric 22:

Health Outcomes Unrelated to Exposure

High

x 1

1

The study report indicated that dosed animals
showed pneumonia more frequently than controls.
The study authors suggested that the development
of pneumonia in rats may have been related to the
prevalence of nasal carcinomas.

Domain 7: Data Presentation and Analysis









Metric 23:

Statistical Methods

Medium

x 1

2

Procedures used for statistical analyses were de-
scribed in detail, and appear to be relevant for
some endpoints (i.e. cancer; the focus of this study).
Owing to differences in the timing of dosing, car-
cinogenicity data for high-dose male rats were com-
pared to controls only (and not to low-dose males).
Statistical analyses for some endpoints (e.g. mor-
tality) appear to consider all groups of male rats,
even though dosing was not concurrent. Incidences
of non-neoplastic lesions were not subjected to sta-
tistical analyses.

Metric 24:

Reporting of Data

High

x 2

2

Data for relevant outcomes (carcinogenicity data)
were provided by exposure group and sex. Data for
other endpoints (e.g. mortality, water consumption)
were not adequately reported.

Overall Quality Determination1"

Unacceptable**

1.9

Extracted

No





* Consistent with our Application of Systematic Review in TSCARisk Evaluations document, if a metric for a data source receives a score of Unacceptable (score = 4),
EPA will determine the study to be unacceptable. In this case, one or more of the metrics were rated as unacceptable. As such, the study is considered unacceptable and
the score is presented solely to increase transparency.

MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; X MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 56 of 187

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Table 18: Animal toxicity evaluation results of NCI et al 1978 for a cancer bioassay- female rats and male and female mice study
on cancer outcomes

Study Citation: NCI (1978). Bioassay of 1,4-dioxane for possible carcinogenicity
Data Type: Cancer bioassay- female rats and male and female mice

HERO ID: 62935

Domain

Metric

Rating^ MWF* Score

Comments^

Domain 1: Test Substance
Metric 1:

Metric 2:

Test Substance Identity
Test Substance Source

Metric 3: Test Substance Purity

Higll X 2 2 The test substance was identified by name and

CASRN.

Higll X 1 1 The source of the test substance was reported, in-

cluding lot numbers. The test substance (one of two
lots) was analyzed to confirm identity and purity
(using vapor phase chromatography and spectrome-
try).

High X 1 1 The purity (one of two lots) was 99.9%. The test

substance was tested for specific impurities (sodium
diethylthiocarbamate, and peroxide); these impuri-
ties were generally present at 0.001% or less. How-
ever one lot showed peroxide levels of 0.1% after
study completion. This deficiency is not likely to
substantially impact the study results.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

Low

x 2

Metric 5:
Metric 6:

Positive Controls
Randomized Allocation

Not Rated
Low

NA
x 1

NA
3

Matched drinking water control groups were used.
However, groups were not placed on study at the
same time. Control female rats were placed on study
later than other groups (by 5 weeks); it was noted
that groups of mice were placed on study "not more
than 7 weeks apart"). Based on data presented
graphically in the study report, the weights of low-
dose mice differed from the body weights of control
and high-dose animals at study week 0.

Positive control group not indicated by study type.
Animals were assigned to control or dose groups "ac-
cording to a series of random numbers;" there were
deficiencies regarding the allocation method that
may impact the study results (e.g. allocation by
animal number).

Domain 3: Exposure Characterization

Continued on next page . .

Page 57 of 187

-------
. continued from previous page

Study Citation: NCI (1978). Bioassay of 1,4-dioxane for possible carcinogenicity
Data Type:	Cancer bioassay- female rats and male and female mice

HERO ID:	62935

Domain



Metric

Rating^

MWF*

Score

Comments^



Metric 7:

Preparation and Storage of Test Substance

Medium

X

1

2

Test substance preparation and storage conditions
were not reported in exhaustive detail ("dioxane so-
lutions prepared in tap water twice per week and
stored in polyethylene containers"). Test substance
stability was demonstrated via analyses conducted
several months after study completion; however,
data on stability of the test substance under the
conditions of administration (in water) were not pro-
vided.



Metric 8:

Consistency of Exposure Administration

High

X

1

1

Dosed water or tap water was available ad libitum.



Metric 9:

Reporting of Doses/Concentrations

Low

X

2

6

As per applicable guideline, water consumption
should be measured at least weekly for the first 13
weeks and at least monthly thereafter. Although
doses in mg/kg-day were provided, these doses were
based on water consumption determined at intervals
during the second year of the bioassay only (and
using 20% of the animals as a representative sam-
ple). The study report indicates that "there were
wide fluctuations in intake at different time periods
within groups."



Metric 10:

Exposure Frequency and Duration

High

X

1

1

As per applicable guideline, the duration of the
study will normally be 24 and 18 months for rats and
mice, respectively. In this study, rats were dosed for
110 weeks and mice were dosed for 90 weeks.



Metric 11:

Number of Exposure Groups and Dose Spac-

Low

X

1

3

Concentrations were chosen based on the results of





ing









previous studies (by Argus et al. 1965). However,













as per applicable guideline, at least three dose level;s
and a concurrent control should be used (the PECO
statement requires at least 2 dose groups and a con-
trol). The study used two dose groups and a con-
trol. The study report noted that the average daily
intake of the test substance in high-dose male mice
was only slightly higher than that of low-dose mice
(estimated 830 vs. 720 mg/kg-day). The difference
between the low- and high-dose in rats was also not
two-fold (as intended). These factors are likely to
have an impact on the study results.



Metric 12:

Exposure Route and Method

High

X

1

1

The route of exposure was reported (i.e. drinking
water); however, no rationale was provided. The
applicable guideline considers drinking water to be
a valid route of administration.

Domain 4: Test Organism

Continued on next page . ..

Page 58 of 187

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.. . continued from previous page

Study Citation: NCI (1978). Bioassay of 1,4-dioxane for possible carcinogenicity
Data Type:	Cancer bioassay- female rats and male and female mice

HERO ID:	62935

Domain

Metric

Ratingt MWF* Score

Comments^

Metric 13: Test Animal Characteristics

High

x 2

Metric 14: Adequacy and Consistency of Animal Hus- High
bandry Conditions

x 1

Metric 15: Number per Group

Medium

x 1

The test animal species, strain, health status, sex,
age, and body weights at study week 0 (provided
graphically) were reported. Animals were obtained
from a commercial laboratory. These animals were
appropriate models for the evaluation of carcino-
genicity (although not the same rat strain used in
previous studies).

Husbandry conditions (temperature, humidity, light
cycles) were reported, and appear to be adequate
(compared to guideline recommendations;) and the
same for control and dosed groups. The applicable
guideline indicates that animals should be housed
individually or in small groups. The study report
indicates that rats were housed 4 per cage and mice
10 per cage. This is unlikely to have had a substan-
tial impact on results (there were no indications of
injuries or death due to overcrowding).

The number of animals per study group was
lower than the typical number used in carcino-
genicity studies in rats (35/sex/group compared to
50/sex/group recommended by guideline). However,
the study report indicated that animal numbers were
adequate for statistical analyses (related to carcino-
genicity).

Domain 5: Outcome Assessment

Metric 16: Outcome Assessment Methodology

Metric 17: Consistency of Outcome Assessment

Medium

Medium

x 2

x 1

Metric 18: Sampling Adequacy

Medium

x 1

Animals from all dose groups were subjected to gross
and microscopic pathology evaluations. The num-
ber of tissues evaluated was not as comprehensive as
that recommended by guideline, but this deficiency
is not likely to substantially impact the study re-
sults.

Surviving rats and mice were sacrificed at 110-117
and 90-93 weeks, respectively. The tissues from
some animals were not evaluated (particularly in an-
imals that died early). Therefore, the numbers of
animals subjected to histopathological evaluations
(with respect to specific organs or tissues) are not
the same as the number of animals placed on study.
Histopathological examinations were performed on
dosed groups and controls. Although details were
not reported (e.g. the numbers of slides evaluated,
individual animal data available but not provided),
these deficiencies are not likely to substantially im-
pact the study results.

Continued on next page

Page 59 of 187

-------
. continued from previous page

Study Citation: NCI (1978). Bioassay of 1,4-dioxane for possible carcinogenicity
Data Type:	Cancer bioassay- female rats and male and female mice

HERO ID:	62935

Domain Metric

Rating^

MWF*

Score

Comments^

Metric 19: Blinding of Assessors

Not Rated

NA

NA

Blinding not reported, but is not required for initial
histopathology review.

Metric 20: Negative Control Response

High

x 1

1

The biological responses of the negative control
groups were adequate (showing no or low incidences
of lesions).

Domain 6: Confounding / Variable Control









Metric 21: Confounding Variables in Test Design and
Procedures

Low

x 2

6

Doses administered to low- and high-dose groups
of rats and mice were not reflective of the in-
tended doses owing (at least in part) to de-
creased palatability (water consumption data were
not provided). Initial body weights were not ex-
plicitly reported (weights at study week 0 were
shown graphically). Rats and mice were housed
in the same room with rats administered dibenzo-
dioxin, 2,7-dichlorobenzodioxin, and 1,2,3,4,6,7,8,9-
octachlorodibenzodioxin.

Metric 22: Health Outcomes Unrelated to Exposure

High

x 1

1

The study report indicated that dosed animals







showed pneumonia more frequently than controls.
The study authors suggested that the development
of pneumonia in rats may have been related to the
prevalence of nasal carcinomas.

Domain 7: Data Presentation and Analysis









Metric 23: Statistical Methods

Medium

x 1

2

Procedures used for statistical analyses were de-
scribed in detail, and appear to be relevant for some
endpoints (i.e. cancer; the focus of this study). Sta-
tistical analyses for some endpoints (e.g. mortality)
appear to consider all groups of rats and mice, even
when dosing was not necessarily concurrent. Inci-
dences of non-neoplastic lesions were not subjected
to statistical analyses.

Metric 24: Reporting of Data

High

x 2

2

Data for relevant outcomes (carcinogenicity data)
were provided by exposure group and sex. Data for
other endpoints (e.g. mortality, water consumption)
were not adequately reported.

Overall Quality Determination1"

Medium



1.8



Extracted

Yes







Continued on next page . ..

Page 60 of 187

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... continued from previous page

Study Citation:

NCI (1978). Bioassay of 1,4-dioxane for possible carcinogenicity





Data Type:

Cancer bioassay- female rats and male and female mice





HERO ID:

62935





Domain

Metric Rating^

MWF* Score

Comments^

MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

if any metric is Unacceptable

Overall rating =

J]. (Metric Score; x MWF;) / J] . MWFj

(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 61 of 187

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Table 19: Animal toxicity evaluation results of Torkelson et al 1974 for a chronic toxicity/carcinogenicity assay in rats study on
mortality, body weight, hematological and immune, clinical chemistry/biochemical, cancer outcomes

II. Results of a 2-year inhalation study in

Study Citation: Torkelson, TR; Leong, BKJ; Kociba, RJ; Richter, WA; Gehring, PJ (1974). 1,4-Dioxane.

rats Toxicology and Applied Pharmacology, 30(2), 287-298
Data Type: Chronic toxicity/carcinogenicity assay in rats

HERO ID: 94807

Domain

Metric

Rating^

MWF* Score

Comments^

Domain 1: Test Substance
Metric 1:
Metric 2:

Test Substance Identity
Test Substance Source

Metric 3: Test Substance Purity

High
Medium

High

x 2
x 1

x 1

2 The test substance was clearly identified by name.

2 The source of the test substance was reported. De-

tails regarding analytical verification of test sub-
stance identity were not provided, but are not likely
to impact the study results.

1 The test substance purity was reportedly 99.9%;

therefore, any effects observed are likely due to the
nominal test substance.

Domain 2: Test Design

Metric 4: Negative and Vehicle Controls

Metric 5: Positive Controls

Metric 6: Randomized Allocation

High X 2 2 The study authors reported using an appropriate

concurrent negative control group (rats exposed to
filtered air only).

Not Rated NA NA Positive controls not indicated by study type.

Low X 1 3 The study authors did not indicate how animals were

allocated to study groups,

Domain 3: Exposure Characterization

Metric 7: Preparation and Storage of Test Substance

Medium

x 1

Metric 8: Consistency of Exposure Administration Medium

Metric 9: Reporting of Doses/Concentrations

High

x 1

x 2

Samples of the test substance were padded with ni-
trogen and stored in bottles until opened for use;
once opened the test substance was used within one
week. The methods and general types of equipment
used to generate the test substance as a vapor were
reported (without detail); this is not likely to impact
the study results.

Details of exposure administration were generally re-
ported (same exposure frequency, consistent cham-
ber design). There were 4 animals per cage during
and in between exposures; time of day of exposures
occurred was not specified.

Analytical, nominal, and target concentrations were
reported. The actual concentration did not deviate
widely (within 10%). The target concentration was
0.36 mg/L; the actual concentration was 0.4 mg/L
(obtained from repeated infared spectrometric anal-
yses).

Continued on next page

Page 62 of 187

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.. . continued from previous page

Study Citation: Torkelson, TR; Leong, BKJ; Kociba, RJ; Richter, WA; Gehring, PJ (1974). 1,4-Dioxane. II. Results of a 2-year inhalation study in

rats Toxicology and Applied Pharmacology, 30(2), 287-298
Data Type: Chronic toxicity/carcinogenicity assay in rats

HERO ID: 94807

Domain Metric Rating^ MWF* Score Comments^

Metric 10:

Exposure Frequency and Duration

High

Exposure frequency and duration were suited to the
study type and outcome of interest.

Metric 11:

Number of Exposure Groups and Dose Spac-

Unacceptable

The dose groups and spacing are not relevant for as-

ing

sessment. As per applicable guideline, there should

be 3 dose groups and a control; the PECO statement
specifies the need for two dose groups and a con-
trol. This study used one group exposed to the test
substance and a control group. The number of ex-
posure groups is not adequate to evaluate exposure-
response relationships. The concentration of the test
substance used in the study was based on the thresh-
old limit value (ACGIH), but was not high enough
to elicit toxicity.

Metric 12:

Exposure Route and Method

High

Rats were exposed to the test substance under dy-
namic exposure conditions.

Domain 4: Test Organism

Metric 13:

Test Animal Characteristics

Medium

General information regarding test animal charac-
teristics (age, health status) were not reported, but
are unlikely to impact the study results. The test
animal species, strain, and sex were reported. Mean
body weights at month 0 of the experiment are
shown graphically in the study report.

Metric 14:

Adequacy and Consistency of Animal Hus-

Low

Husbandry conditions were not reported in suffi-

bandry Conditions

cient detail to determine if conditions were the

same/adequate between control and exposed groups.

Metric 15:

Number per Group

High

The number of animals per groups was reported
and adequate for the study type. Typically
50/sex/group are used for rodent cancer bioassays;
this study used 288 rats/sex/exposure group and 192
rats/sex/group for controls.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

The outcome methodology addressed the intended
outcomes of interest.

Metric 17:

Consistency of Outcome Assessment

High

Outcomes appear to have been assessed consistently
across groups (same time after initial exposure) and
using the same protocols.

Metric 18:

Sampling Adequacy

High

Endpoints (including hematology and clinical chem-
istry, gross and microscopic pathology) were evalu-
ated in all surviving animals.

Continued on

next page . ..

Page 63 of 187

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. continued from previous page

II. Results of a 2-year inhalation study in

Study Citation: Torkelson, TR; Leong, BKJ; Kociba, RJ; Richter, WA; Gehring, PJ (1974). 1,4-Dioxane.

rats Toxicology and Applied Pharmacology, 30(2), 287-298
Data Type:	Chronic toxicity/carcinogenicity assay in rats

HERO ID:	94807

Domain

Metric

Rating^ MWF* Score

Comments^

Metric 19:

Blinding of Assessors

Not Rated

NA

NA

Blinding not required for initial histopathology ex-
aminations (other endpoints evaluated were not sub-
jective).

Metric 20:

Negative Control Response

High

x 1

1

In general, the incidence of tumors in control and ex-
posed rats was low or none. Both treated rats and
controls showed reticulum cell sarcomas and mam-
mary tumors. The study authors indicated that "nu-
merous tumors characteristic of this strain were seen
in all groups."

Domain 6: Confounding / Variable Control

Metric 21: Confounding Variables in Test Design and Medium
Procedures

Metric 22: Health Outcomes Unrelated to Exposure

High

x 2

x 1

Initial body weights were not explicitly specified
(body weights at month 0 of treatment were shown
graphically). No information on respiratory rate was
reported, but this is not expected to substantially
impact the study results.

Data on attrition and/or health outcomes not re-
lated to exposure were not reported because there
were not any significant differences among groups.

Domain 7: Data Presentation and Analysis

Metric 23: Statistical Methods

Metric 24: Reporting of Data

High
Medium

x 1

x 2

Statistical methods were described (in minimal de-
tail) and appear to be appropriate.

Data for all outcomes were presented by exposure
group and sex. Measures of variation were not shown
for all endpoints (hematology and clinical chemistry
parameters).

Overall Quality Determination1"

Unacceptable**

1.6

Extracted

No





* Consistent with our Application of Systematic Review in TSCARisk Evaluations document, if a metric for a data source receives a score of Unacceptable (score = 4),
EPA will determine the study to be unacceptable. In this case, one or more of the metrics were rated as unacceptable. As such, the study is considered unacceptable and
the score is presented solely to increase transparency.

MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; X MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 64 of 187

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Table 20: Animal toxicity evaluation results of Kasai et al 2009 for a 2-year cancer bioassay study on cancer, mortality, hepatic,
renal, respiratory, hematological and immune, clinical chemistry/biochemical, nutrition and metabolic/adult exposure body weight,
reproductive outcomes

Study Citation: Kasai, T; Kano, H; Umeda, Y; Sasaki, T; Ikawa, N; Nishizawa, T; Nagano, K; Arito, H; Nagashima, H; Fukushima, S (2009). Two-year

inhalation study of carcinogenicity and chronic toxicity of 1,4-dioxane in male rats Inhalation Toxicology, 21(11), 889-897
Data Type: 2-year cancer bioassay

HERO ID: 193803

Domain

Metric

Rating^ MWF* Score

Comments^

Domain 1: Test Substance
Metric 1:
Metric 2:

Test Substance Identity
Test Substance Source

Metric 3: Test Substance Purity

High X 2 2 The test substance was identified definitively.

High X 1 1 The source of the test substance was reported, in-

cluding manufacturer, and its identity was verified
by analytical methods.

Medium X 1 2 The test chemical was reported as reagent grade

(greater than 99% pure) and purity was also eval-
uated by the laboratory via gas chromatography-
mass spectrometry (GC-MS). I downgraded this to
medium because all seven lots tested were found to
contain butylhydroxytoluene (avg level of 4.6 ppm
[w/w]) by GC-MS, although no peak corresponding
to this substance was found in air samples collected
from the inhalation chamber.

Domain 2: Test Design

Metric 4: Negative and Vehicle Controls

Metric 5: Positive Controls

Metric 6: Randomized Allocation

Higll X 2 2 The study authors reported using an appropriate

concurrent negative control group.

Not Rated NA NA Not applicable - Positive control group is not indi-

cated by study type.

High X 1 1 The animals were divided by stratified randomiza-

tion into body weight-matched groups.

Domain 3: Exposure Characterization

Metric 7: Preparation and Storage of Test Substance High x 1

Metric 8: Consistency of Exposure Administration Medium x 1

The test substance was found to be stable through-
out the 7-month period of storage, as determined by
gas chromatography. The methods and equipment
used to generate the test substance were appropri-
ate.

Details of exposure administration were reported
and were consistent among the groups. However, I
downgraded this to medium because the report does
not specifically state that exposures occurred at the
same time of day for all animals.

Continued on next page . .

Page 65 of 187

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. continued from previous page

Study Citation: Kasai, T; Kano, H; Umeda, Y; Sasaki, T; Ikawa, N; Nishizawa, T; Nagano, K; Arito, H; Nagashima, H; Fukushima, S (2009). Two-year

inhalation study of carcinogenicity and chronic toxicity of 1,4-dioxane in male rats Inhalation Toxicology, 21(11), 889-897
Data Type: 2-year cancer bioassay

HERO ID: 193803

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 9:

Reporting of Doses/Concentrations

High

x 2

Actual vapor concentrations in the exposure cham-
bers were measured and mean concentrations over
the exposure period were reported (shown in Figure
1 of the study report).

Metric 10:

Exposure Frequency and Duration

High

x 1

The exposure frequency and duration of exposure
were reported and were appropriate for this type of
study.

Metric 11:

Number of Exposure Groups and Dose Spac-

High

x 1

The number of exposure groups and concentration

ing

spacing were justified and adequate for the purpose

of this study.

Metric 12:

Exposure Route and Method

High

x 1

The route and method of exposure were reported
and suited to the test substance. The number of air
changes per hour was adequate (12/hour).

Domain 4: Test Organism

Metric 13: Test Animal Characteristics

Medium

Metric 14: Adequacy and Consistency of Animal Hus- High
bandry Conditions

Metric 15: Number per Group

High

x 2

x 1

Most of the test animal characteristics were reported
(species, strain, sex, age, starting body weight);
however, health status at the start of the study was
not reported.

All husbandry conditions were reported and were ad-
equate and consistent among the groups and con-
trols.

The number of animals per study group was reported
and appropriate for the study type.

Domain 5: Outcome Assessment

Metric 16: Outcome Assessment Methodology

Metric 17: Consistency of Outcome Assessment

Metric 18: Sampling Adequacy

High

Low

High

x 2

x 1

The outcome assessment methodology addressed the
intended outcomes of interest and was sensitive for
the outcomes of interest.

The outcome assessment protocol was reported;
however, the descriptions of each outcome method-
ology do not specifically state that some outcomes
(e.g., urine, blood) were sampled at the same
time/day for all groups.

Details regarding sampling for the outcomes of in-
terest were reported and the study used adequate
sampling for the outcomes (e.g., number of animals
per group was adequate for the study type).

Continued on next page

Page 66 of 187

-------
. continued from previous page

Study Citation: Kasai, T; Kano, H; Umeda, Y; Sasaki, T; Ikawa, N; Nishizawa, T; Nagano, K; Arito, H; Nagashima, H; Fukushima, S (2009). Two-year

inhalation study of carcinogenicity and chronic toxicity of 1,4-dioxane in male rats Inhalation Toxicology, 21(11), 889-897
Data Type:	2-year cancer bioassay

HERO ID:	193803

Domain Metric

Rating^

MWF*

Score

Comments^

Metric 19: Blinding of Assessors

Not Rated

NA

NA

No subjective outcomes to which blinding would be









required were included and automated techniques









(e.g., for blood biochemical analysis) were used for









blood biochemical analysis. Histopathology exami-









nation results were not described as a re-evaluation









so I considered this metric N/A.

Metric 20: Negative Control Response

High

x 1

1

The negative control response was adequate.

Domain 6: Confounding / Variable Control









Metric 21: Confounding Variables in Test Design and

Low

x 2

6

There were no reported differences in initial weight,

Procedures







or food or water intake. However, this substance is







considered an irritant (addressed in Discussion on









p. 895, e.g., see citation Boatman & Knaak, 2001);









however, respiratory rate measurement was not re-









ported and this study, so I downgraded this metric









rating to Low.

Metric 22: Health Outcomes Unrelated to Exposure

High

x 1

1

No indications of attrition or health outcomes unre-









lated to exposure.

Domain 7: Data Presentation and Analysis









Metric 23: Statistical Methods

High

x 1

1

The statistical methods were clearly described and







appropriate for the data set.

Metric 24: Reporting of Data

Medium

x 2

4

Data for exposure-related findings were shown for









each exposure group. However, severity scores were









not presented for histopathological changes that









were observed in this study (e.g., pre- and non-









neoplastic changes in Table 3) so I downgraded the









score to medium.

Overall Quality Determination1"

High



1.4



Extracted

Yes









MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; x MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 67 of 187

-------
Table 21: Animal toxicity evaluation results of Argus et al 1973 for a carcinogenicity-liver (dose response), electron microscopy
study on cancer outcomes

Study Citation: Argus, MF; Sohal, RS; Bryant, GM; Hoch-Ligeti, C; Arcos, JC (1973). Dose-response and ultrastructural alterations in dioxane

carcinogenesis. Influence of methylcliolantlirene on acute toxicity European Journal of Cancer, 9(4), 237-243
Data Type: Carcinogenicity-liver (dose response), electron microscopy

HERO ID: 62912

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

Medium

x 2

Identified by name and source same as Argus et al.,

1965 , which limits uncertainties

Metric 2:

Test Substance Source

Medium

x 1

Source reported but no additional details were re-

ported

Metric 3:

Test Substance Purity

Low

x 1

Purity was not reported

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

x 2

There were no apparent differences in the concurrent

control group.

Metric 5:

Positive Controls

Not Rated

This metric was not applicable.

Metric 6:

Randomized Allocation

Low

x 1

The study did not report how animals were allocated

to study groups.

Domain 3: Exposure Characterization

Metric 7:

Preparation and Storage of Test Substance

High

x 1

Solutions were prepared fresh daily in drinking wa-

Metric 8:

Consistency of Exposure Administration

High

X 1

There were no apparent inconsistencies in exposure

administration.

Metric 9:

Reporting of Doses/Concentrations

High

x 2

The doses were reported along with average fluid

consumption

Metric 10:

Exposure Frequency and Duration

High

x 1

Duration was provided

Metric 11:

Number of Exposure Groups and Dose Spac-

High

x 1

The number of exposure groups and dose spacing

ing

were appropriate

Metric 12:

Exposure Route and Method

High

x 1

The route and method were appropriate.

Domain 4: Test Organism

Metric 13:

Test Animal Characteristics

Medium

x 2

The species, strain, sex, age, initial body weight

range, and source were reported. The health status

of the animals was not reported.

Metric 14:

Adequacy and Consistency of Animal Hus-

Low

x 1

Husbandry conditions were not sufficiently reported

bandry Conditions

to evaluate if adequate.

Continued on next page . . .

Page 68 of 187

-------
. continued from previous page

Study Citation: Argus, MF; Sohal, RS; Bryant, GM; Hoch-Ligeti, C; Arcos, JC (1973). Dose-response and ultrastructural alterations in dioxane

carcinogenesis. Influence of methylcholanthrene on acute toxicity European Journal of Cancer, 9(4), 237-243
Data Type:	Carcinogenicity-liver (dose response), electron microscopy

HERO ID:	62912

Domain Metric

Rating^

MWF*

Score

Comments^

Metric 15: Number per Group

Medium

X 1

2

The reported number of animals ranged from 28 to
32, but the group(s) that had less than 30 animals
(slightly lower than cancer bioassay) was not speci-
fied.

Domain 5: Outcome Assessment

Metric 16: Outcome Assessment Methodology

Metric 17: Consistency of Outcome Assessment

Medium
High

x 2
x 1

4
1

Limited details in outcome assessment methodology
was provided.

It is inferred that outcome assessment was consis-
tent.

All animals were assessed.

This metric is not applicable.

The biological responses of the control animals in
the dose response study were not reported.

Metric 18: Sampling Adequacy
Metric 19: Blinding of Assessors
Metric 20: Negative Control Response

High

Not Rated
Unacceptable

x 1
NA
x 1

1

NA
4

Domain 6: Confounding / Variable Control

Metric 21: Confounding Variables in Test Design and
Procedures

Metric 22: Health Outcomes Unrelated to Exposure

High
Low

x 2
x 1

2

3

No differences were reported.
Details were not reported

Domain 7: Data Presentation and Analysis

Metric 23: Statistical Methods
Metric 24: Reporting of Data

Low
Low

x 1
x 2

3
6

Statistical methods were not reported

Data were described in the text, and descriptive tu-
mor characteristics were not distinguished among
groups. Effective tumor doses were reported

Overall Quality Determination1"

Unacceptable**

—> Low§





Extracted

Yes









* Consistent with our Application of Systematic Review in TSCARisk Evaluations document, if a metric for a data source receives a score of Unacceptable (score = 4), EPA
will determine the study to be unacceptable. In this case, one or more of the metrics were rated as unacceptable. As such, the study is considered unacceptable and the score
is presented solely to increase transparency.

MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

if any metric is Unacceptable

Overall rating =

]T\ (Metric Score; x MWF;) / . MWFj

(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed
out and an arrow points to the new rating,
ft This metric met the criteria for high confidence as expected for this type of study

§ Evaluator's explanation for rating change: "The study would be upgraded because a description of the tumors observed was provided which is informative. Also, effective
tumor doses were provided."

Page 69 of 187

-------
Table 22: Animal toxicity evaluation results of Jbrc et al 1998 for a cancer bioassay and non-neoplastic lesions study on cancer,
renal, hepatic, respiratory outcomes

Study Citation: JBRC (1998). Two-year studies of 1,4-dioxane in F344 rats and BDF1 mice (drinking water)
Data Type:	Cancer bioassay and non-neoplastic lesions

HERO ID:	196240

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance













Metric 1:

Test Substance Identity

High

X

2

2

Identified by name, structure, and CASRN

Metric 2:

Test Substance Source

Medium

X

1

2

Source was reported but no additional information.

Metric 3:

Test Substance Purity

High

X

1

1

Purity such that effects likely due to test substance

Domain 2: Test Design













Metric 4:

Negative and Vehicle Controls

High

X

2

2

Appropriate negative control group was included

Metric 5:

Positive Controls

Not Rated

NA

NA

Not applicable for this study

Metric 6:

Randomized Allocation

Low

X

1

3

Allocation of animals was not reported

Domain 3: Exposure Characterization











Metric 7:

Preparation and Storage of Test Substance

Low

X

1

3

Test substance was administered in the drinking wa-













ter, but additional details were not reported..

Metric 8:

Consistency of Exposure Administration

High

X

1

1

Exposures were consistent

Metric 9:

Reporting of Doses/Concentrations

High

X

2

2



Metric 10

Exposure Frequency and Duration

High

X

1

1



Metric 11

Number of Exposure Groups and Dose Spac-

High

X

1

1



Metric 12

ing

Exposure Route and Method

High

X

1

1



Domain 4: Test Organism













Metric 13

Test Animal Characteristics

Medium

X

2

4

The source, species, strain, sex, and age were re-













ported. Starting body weight and health status were













not reported

Metric 14

Adequacy and Consistency of Animal Hus-

High

X

1

1

All husbandry conditions were reported.



bandry Conditions











Metric 15

Number per Group

High

X

1

1



Domain 5: Outcome Assessment











Metric 16

Outcome Assessment Methodology

High

X

2

2

Outcome methodology was appropriate and sensitive

Metric 17

Consistency of Outcome Assessment

High

X

1

1

Outcomes were assessed consistently

Metric 18

Sampling Adequacy

High

X

1

1

Sampling was appropriate

Metric 19

Blinding of Assessors

Not Rated

NA

NA

Not applicable for this study

Metric 20

Negative Control Response

High

X

1

1



Domain 6: Confounding / Variable Control

Continued on next page . ..

Page 70 of 187

-------
... continued from previous page

Study Citation:
Data Type:
HERO ID:

JBRC (1998). Two-year studies of 1,4-dioxane in F344 rats and BDF1

Cancer bioassay and non-neoplastic lesions

196240

mice (drinking water)

Domain

Metric

Rating^

MWF*

Score

Comments^



Metric 21: Confounding Variables in Test Design and
Procedures

Metric 22: Health Outcomes Unrelated to Exposure

High
High

x 2
x 1

2
1

There were no differences among groups unrelated
to exposure

Domain 7: Data Presentation and Analysis

Metric 23: Statistical Methods
Metric 24: Reporting of Data

High
High

x 1
x 2

1

2

Statistical analyses were reported and appropriate
Outcomes were reported.

Overall Quality Determination1"

High



1.2



Extracted



Yes









MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; x MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating,
ft This metric met the criteria for high confidence as expected for this type of study

Page 71 of 187

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Table 23: Animal toxicity evaluation results of Kano et al 2009 for a 2-year cancer bioassay study on cancer outcomes

Study Citation: Kano, H; Umeda, Y; Kasai, T; Sasaki, T; Matsumoto, M; Yamazaki, K; Nagano, K; Arito, H; Fukushima, S (2009). Carcinogenicity

studies of 1,4-dioxane administered in drinking-water to rats and mice for 2 years Food and Chemical Toxicology, 47(11), 2776-2784
Data Type: 2-year cancer bioassay

HERO ID: 594539

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Identified by CASRN and each lot analyzed by IR

and GC.

Metric 2:

Test Substance Source

High

Obtained from manufacturer.

Metric 3:

Test Substance Purity

High

>99% pure; confirmed by GC

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

Adequately reported

Metric 5:

Positive Controls

Not Rated

Not indicated for study type.

Metric 6:

Randomized Allocation

High

Stratified randomization; matched by body weight

Domain 3: Exposure Characterization

Metric 7:

Preparation and Storage of Test Substance

High

Adequately reported; prepared twice per week and

stable at 4 days post-preparation.

Metric 8:

Consistency of Exposure Administration

High

Drinking water available to all animals ad libitum

Metric 9:

Reporting of Doses/Concentrations

High

Data provided on water consumption; no difference

across groups.

Metric 10:

Exposure Frequency and Duration

High

Consistent with test guideline for study type.

Metric 11:

Number of Exposure Groups and Dose Spac-

High

Highest dose chosen so as not to exceed the MTD.

Metric 12:

ing

Exposure Route and Method

High

Adequately reported. Consistent with test guideline

for study type.

Domain 4: Test Organism

Metric 13:

Test Animal Characteristics

High

Adequately reported. Consistent with test guide-

lines for study type.

Metric 14:

Adequacy and Consistency of Animal Hus-

High

Adequately reported. Consistent with test guide-

bandry Conditions

lines for study type.

Metric 15:

Number per Group

High

50/sex/group; consistent with test guidelines for

study type.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

Consistent with test guidelines for study type.

Metric 17:

Consistency of Outcome Assessment

High

No anomalies reported.

Metric 18:

Sampling Adequacy

High

Consistent with test guidelines for study type.

Metric 19:

Blinding of Assessors

Low

Not addressed.

Continued on

next page . .

Page 72 of 187

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... continued from previous page

Study Citation: Kano, H; Umeda, Y; Kasai, T; Sasaki, T; Matsumoto, M; Yamazaki, K; Nagano, K; Arito, H; Fukushima, S (2009). Carcinogenicity

studies of 1,4-dioxane administered in drinking-water to rats and mice for 2 years Food and Chemical Toxicology, 47(11), 2776-2784
Data Type:	2-year cancer bioassay

HERO ID:	594539

Domain Metric

Rating^

MWF*

Score

Comments^

Metric 20: Negative Control Response

High

X 1

1

Adequately reported; no unusual results.

Domain 6: Confounding / Variable Control









Metric 21: Confounding Variables in Test Design and

High

x 2

2

Body-weight matching; no difference in food/water

Procedures







consumption.

Metric 22: Health Outcomes Unrelated to Exposure

High

x 1

1

Attrition was related to exposure.

Domain 7: Data Presentation and Analysis









Metric 23: Statistical Methods

High

x 1

1

Appropriate methods chosen; adequately reported.

Metric 24: Reporting of Data

High

x 2

2

Multiple data tables summarize all endpoints.

Overall Quality Determination1"

High



1.1



Extracted

Yes









MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

J]. (Metric Score; x MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 73 of 187

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5 Developmental

Table 24: Animal toxicity evaluation results of Giavini et al 1985 for a developmental-fetal effects study on growth (early life) and
development outcomes

Study Citation: Giavini, E; Vismara, C; Broccia, ML (1985). Teratogenesis study of dioxane in rats Toxicology Letters, 26(1), 85-88

Data Type: Developmental-fetal effects

HERO ID: 62924

Domain Metric Rating^ MWF* Score Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

Low

The test substance was identified by name only

Metric 2:

Test Substance Source

Medium

Source identified but no other details were reported.
The omitted details are unlikely to have a substan-
tial impact on results.

Metric 3:

Test Substance Purity

High

Purity and impurity identified; purity such that ef-
fects due to test substance.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

Appropriate controls used.

Metric 5:

Positive Controls

Not Rated

This metric is not applicable.

Metric 6:

Randomized Allocation

Low

The method of allocation was not reported.

Domain 3: Exposure Characterization

Metric 7:

Preparation and Storage of Test Substance

Medium

Limited details on preparation and no details on
storage were reported.

Metric 8:

Consistency of Exposure Administration

High

Exposures administered consistently

Metric 9:

Reporting of Doses/Concentrations

High

Doses were reported without ambiguity.

Metric 10:

Exposure Frequency and Duration

High

Details were reported and appropriate.

Metric 11:

Number of Exposure Groups and Dose Spac-
ing

High

Number of exposure groups and spacing were appro-
priate

Metric 12:

Exposure Route and Method

High

The route and method were suited to the test sub-
stance.

Domain 4: Test Organism

Metric 13:

Test Animal Characteristics

Medium

The source, species, strain, initial body weight, and
sex were reported. The age and health status were
not reported.

Metric 14:

Adequacy and Consistency of Animal Hus-

Medium

The humidity, light-dark cycle,, temperature, and

bandry Conditions

availability of food and water were reported. The

number of animals/cage was not reported.

Metric 15:

Number per Group

Medium

The total number of animals per group were differ-
ent, but a sufficient number of animals were available
for statistical analysis.

Domain 5: Outcome Assessment

Continued on next page . ..

Page 74 of 187

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. continued from previous page

Study Citation: Giavini, E; Vismara, C; Broccia, ML (1985). Teratogenesis study of dioxane in rats Toxicology Letters, 26(1), 85-£
Data Type:	Developmental-fetal effects

HERO ID:	62924

Domain

Metric

Rating^ MWF* Score

Comments^

Metric 16:

Outcome Assessment Methodology

High

X

2

2

Outcome assessment methodology was appropriate
and sensitive.

Metric 17:

Consistency of Outcome Assessment

High

X

1

1

Outcomes were assessed consistently.

Metric 18:

Sampling Adequacy

High

X

1

1

Sampling was adequate for the outcomes of interest.

Metric 19:

Blinding of Assessors

Not Rated

NA

NA

This metric was not applicable.

Metric 20:

Negative Control Response

High

X

1

1

There were no apparent issues with the biological
response of the negative control group.

Domain 6: Confounding / Variable Control











Metric 21:

Confounding Variables in Test Design and
Procedures

Medium

X

2

4

There were reported differences in maternal food
consumption and body weight gain associated with
treatment

Metric 22:

Health Outcomes Unrelated to Exposure

High

X

1

1

No health outcomes unrelated to exposure were re-











ported or could be inferred .

Domain 7: Data Presentation and Analysis











Metric 23:

Statistical Methods

Medium

X

1

2

Statistical tests were reported, but the parameters
to which they were applied were not reported.

Metric 24:

Reporting of Data

High

X

2

2

Data were presented for all outcomes by exposure
groups.

Overall Quality Determination



High





1.5



Extracted



Yes









MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; x MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 75 of 187

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6 Genetic toxicity studies

Table 25: Animal toxicity evaluation results of Goldsworthy et al 1991 for nasal epithelium DNA repair in rats

Study Citation: T. L. Goldsworthy, T. M. Monticello, K. T. Morgan, E. Bermudez, D. M. Wilson, R. Jackh,Butterworth BE (1991). Examination of
potential mechanisms of carcinogenicity of 1,4-dioxane in rat nasal epithelial cells and hepatocytes Archives of Toxicology, 65(1,1), 1-9
Data Type: Nasal Epithelium DNA repair

HERO ID: 62925

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Test substance identified as "1,4-dioxane".

Metric 2:

Test Substance Source

High

The source of the test substance was reported. The

batch/lot number was not reported, but the test sub-

stance is not expected to vary in composition.

Metric 3:

Test Substance Purity

High

Test substance was reported to be of HPLC grade,

99.9% purity.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

Concurrent negative controls were utilized (water).

Metric 5:

Positive Controls

High

A positive control was utilized.

Metric 6:

Randomized Allocation

Low

The study did not report how animals were allocated

to study groups.

Domain 3: Exposure Characterization

Metric 7:

Preparation and Storage of Test Substance

Medium

Preparation and storage of the test substance was

not reported, but test substance administered in wa-

ter and test substance is known to be soluble in wa-

Metric 8:

Consistency of Exposure Administration

High

A consistent concentration was administered in

drinking water and gavage treatment was conducted

consistently across groups.

Metric 9:

Reporting of Doses/Concentrations

Low

Concentration (single concentration) administered

in drinking water was reported. Additional single

gavage doses were reported. No palatability issues

were described, but body weights and water con-

sumption were not reported.

Metric 10:

Exposure Frequency and Duration

High

The exposure frequency and duration was reported

and appropriate for this endpoint.

Metric 11:

Number of Exposure Groups and Dose Spac-

Medium

Number of exposure groups and spacing of exposure

irip-

levels were adequate to show results relevant to the

1A16

outcome of interest, but there was no justification

for why the doses and spacing were selected.

Metric 12:

Exposure Route and Method

High

The route of exposure was appropriate for this end-

point.

Domain 4: Test Organism

Continued on next page . ..

Page 76 of 187

-------
.. . continued from previous page

HERO ID: 62925

Domain

Metric

Rating^

MWF*

Score

('o m me ill srr

Metric 13:

Test Animal Characteristics

High

x 2

Test animal characteristics were reported.

Metric 14:

Adequacy and Consistency of Animal Hus-

High

x 1

Animal husbandry conditions were adequate and

bandry Conditions

consistent across control and exposed groups.

Metric 15:

Number per Group

Unacceptable

x 1

The number of animals per group is not specifically

reported, but the footnote of Table 6 suggests that

only two animals were used.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

x 2

The outcome assessment methodology was appropri-

ate for this endpoint.

Metric 17:

Consistency of Outcome Assessment

High

x 1

The outcome assessment was carried out consis-

tently across study groups.

Metric 18:

Sampling Adequacy

High

x 1

An adequate number of slides and cells were evalu-

Metric 19:

Blinding of Assessors

Not Rated

Blinding is not a concern in this study.

Metric 20:

Negative Control Response

High

x 1

The control response was adequate.

Domain 6: Confounding / Variable Control

Metric 21:

Confounding Variables in Test Design and

Low

x 2

The study did not report on initial body weights or

Procedures

food/water intake during this drinking water study.

Metric 22:

Health Outcomes Unrelated to Exposure

High

x 1

No health outcomes or deaths were reported in the

study.

Domain 7: Data Presentation and Analysis

Metric 23:

Statistical Methods

High

x 1

Statistical methods were reported and appropriate

for the dataset.

Metric 24:

Reporting of Data

High

x 2

Data were reported for all outcomes and groups.

Overall Quality Determination1" Unacceptable** 1.5

Extracted No

* Consistent with our Application of Systematic Review in TSCARisk Evaluations document, if a metric for a data source receives a score of Unacceptable (score = 4),
EPA will determine the study to be unacceptable. In this case, one or more of the metrics were rated as unacceptable. As such, the study is considered unacceptable and
the score is presented solely to increase transparency.

MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

if any metric is Unacceptable

Overall rating =

J]. (Metric Score; x MWF;) / J] . MWFj

(round to the nearest tenth) otherwise

Page 77 of 187

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Table 26: Animal toxicity evaluation results of Kitchin and Brown 1990 for acute rats study on liver DNA damage

Study Citation: K. T. Kitchin, J. L. Brown (1990). Is 1,4-dioxane a genotoxic carcinogen? Cancer Letters, 53(1,1), 67-71
Data Type: Acute rat liver DNA damage

HERO ID: 62928

Domain

Metric

Ratingt MWF* Score

Comments^

Domain 1: Test Substance

Metric 1: Test Substance Identity

Metric 2: Test Substance Source

Metric 3: Test Substance Purity

High
High

High

X 2 2 The test substance was identified by name: 1,4 diox-

ane, and mol wt.: 88.11

X 1 1 Test substance was obtained from Aldrich Chem Co.

Inc. Milwaukee, WI. No information reported on
batch/lot number; however, the test substance is un-
likely to vary in composition.

X 1 1 The test substance was highly pure: 99+% purity,

no impurities were reported.

Domain 2: Test Design

Metric 4: Negative and Vehicle Controls

Metric 5: Positive Controls
Metric 6: Randomized Allocation

Low

Not Rated
Low

x 2

NA
x 1

NA
3

A corn oil control group was indicated in Table 1;
however, the paper does not explicitly state that the
test material was dissolved in corn oil or whether the
corn oil controls were administered the same dose
volume at the same time prior to sacrifice (this was
assumed).

NA: positive control was not necessary based on
study type

Allocation of animals into study groups is not re-
ported

Domain 3: Exposure Characterization

Metric 7: Preparation and Storage of Test Substance

Low

Metric 8: Consistency of Exposure Administration Low

Metric 9: Reporting of Doses/Concentrations

Medium

x 1

x 2

Table 1 suggests that the test substance was pre-
pared in corn oil (not explicitly stated). Test sub-
stance storage was not described; however, omission
of these details are unlikely to have a substantial
impact on results (only 2 doses were given 17 hours
apart).

Table 1 states oral admin., and indicates that dos-
ing was administered 4 and 21 hours prior to sacri-
fice which occurred at consistently at 12:00. These
details suggest a gavage route of exposure; however,
the gavage volume was not reported.

Doses are reported in mg/kg: 0, 168, 840, 2550,
4200 mg/kg. the doses were given according to body
weight (route not specified but assumed to be gav-
age as negative control is corn oil- common use in
gavage and 2 single administrations indicate gavage
as well)

Continued on next page . .

Page 78 of 187

-------
. continued from previous page

Study Citation: K. T. Kitchin, J. L. Brown (1990). Is 1,4-dioxane a genotoxic carcinogen? Cancer Letters, 53(1,1), 67-71
Data Type: Acute rat liver DNA damage

HERO ID: 62928

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 10:

Exposure Frequency and Duration

High

X 1

Dosing was 2 single administrations at 21 and 4
hours prior to sacrifice. Cited previous literature in-
dicating that a 4h timepoint was sufficient for DNA
damage

Metric 11:

Number of Exposure Groups and Dose Spac-
ing

High

X 1

Doses were diluted from the acute rat oral
LD50:100%, 60%, 20%, 4%

Metric 12:

Exposure Route and Method

Low

X 1

Table 1 reports route as oral and the method is
assumed to be gavage (corn oil as vehicle control,
mg/kg dosing administered as single doses (2x),
however it was not reported

Domain 4: Test Organism

Metric 13:

Test Animal Characteristics

Medium

x 2

Female SD rats (CD strain) from Charles river lab-
oratories (Raleigh NC) were 90 days old and accli-
mated for several weeks. Health status and starting
BW was not reported. Animal is routinely used for
outcome of interest

Metric 14:

Adequacy and Consistency of Animal Hus-

High

x 1

72 + /- 4 degrees F, 50 + /-10% humidity and 12 h

bandry Conditions

light cycle 6am-6pm on, housed 3/ cage and accli-

mated for several weeks. Husbandry conditions were
adequate and same for all dose groups

Metric 15:

Number per Group

High

x 1

4-13 F rats/group reported in table 1. The number
of animals per study group was appropriate for the
study type and outcome analysis

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

Medium

x 2

DNA damage was reportedly done "as previously de-
scribed" reference 8. Table 1 indicates that DNA
damage was evaluated by alkaline elution which is a
sensitive and appropriate method for detection of
DNA damage, but few methodological details are
provided

Metric 17:

Consistency of Outcome Assessment

High

x 1

The outcome assessment was consistent in protocol

and time across all study groups

Metric 18:

Sampling Adequacy

Not Rated

Metric 19:

Blinding of Assessors

Not Rated

Details regarding blinding are not applicable for this
study type as assessing subjective outcomes was not
necessary

Metric 20:

Negative Control Response

High

x 1

The biological response in the negative control group
was adequate

Domain 6: Confounding / Variable Control

Continued on next page . ..

Page 79 of 187

-------
. continued from previous page

Study Citation: K. T. Kitchin, J. L. Brown (1990). Is 1,4-dioxane a genotoxic carcinogen? Cancer Letters, 53(1,1), 67-71
Data Type:	Acute rat liver DNA damage

HERO ID:	62928

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 21:

Confounding Variables in Test Design and
Procedures

Low

x 2

6

Initial BW was not reported. The specific route was
not reported (oral not further described) therefore it
is not known if palatability influenced outcome and
it was not reported. No body weight food or water
consumption or clinical signs were reported

Metric 22:

Health Outcomes Unrelated to Exposure

Low

x 1

3

Data on attrition and/or health outcomes unrelated
to exposure were not reported for each study group

Domain 7: Data Presentation and Analysis









Metric 23:

Statistical Methods

High

x 1

1

Statistical tests include an ANOVA followed by a









student's t test of findings from the ANOVA; suffi-
cient data were provided to allow for other statisti-
cal tests..

Metric 24:

Reporting of Data

High

x 2

2

Quantitative data are reported in table 1 as mean
+ /- SEM with n reported below. Reported by dose
group

Overall Quality Determination1"

Medium

1.9

Extracted

Yes





MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; x MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 80 of 187

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Table 27: Animal toxicity evaluation results of Yoon et al 1985 for sex linked recessive lethal mutations in Drosophila study

Study Citation: J. S. Yoon, J. M. Mason, R. Valencia, R. C. Woodruff, S. Zimmering (1985). Chemical mutagenesis testing in Drosophila. IV. Results

of 45 coded compounds tested for the National Toxicology Program Environmental Mutagenesis, 7(3,3), 349-367
Data Type: 1, 4, D sex linked recessive lethal in drosophila

HERO ID: 194373

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

x 2

Table 1 number 24 1,4 dioxane, CASRN: 123-91-1

and structure included, MW was 88.12

Metric 2:

Test Substance Source

High

x 1

Test substance source is Fisher #785133 (in table

1). Lot number was not reported; however, the test

substance is unlikely to vary in composition

Metric 3:

Test Substance Purity

Low

x 1

Test substance purity reported in table 1: Labeled

purity- "purified", analyzed purity- blank

Domain 2: Test Design

Metric 4: Negative and Vehicle Controls

Metric 5: Positive Controls

Metric 6: Randomized Allocation

Medium

Not Rated
Low

x 2

NA
x 1

NA
3

Negative concurrent controls were used. It was not
reported if the negative controls were vehicle or un-
treated

Allocation of animals into study groups is not re-
ported but may be included in the previous papers
cited (Woodruff et al. 1984, Zimmering et al., 1984,
or Valencia et al., 1985) for stock, mating schemes,
protocols and methods.

Domain 3: Exposure Characterization

Metric 7: Preparation and Storage of Test Substance Medium x 1

Metric 8: Consistency of Exposure Administration Not Rated NA

Metric 9: Reporting of Doses/Concentrations Medium x 2

Metric 10: Exposure Frequency and Duration High x 1

Metric 11: Number of Exposure Groups and Dose Spac- Low x 1

ing

2 The test substance was prepared using water as the
solvent, storage were not described but omission of
these details is unlikely to have a substantial impact
on results (3 day diet and injection).

NA Protocols were from previously cited literature and
were not reported in text.

4 Feeding dose reported in table 2 as 0, 35,000 ppm;
injection doses are reported as 0, 50,000 ppm.

1 Feeding study duration was 3 days (assume contin-

uous); while exact doses achieved could not be con-
firmed, injection was administered if no mutation
occurred with dietary exposure

3 Concentration was selected based on solubility,
palatability, and toxicity (not further described).
Single dose group for each route.

Continued on next page ..

Page 81 of 187

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.. . continued from previous page

Study Citation: J. S. Yoon, J. M. Mason, R. Valencia, R. C. Woodruff, S. Zimmering (1985). Chemical mutagenesis testing in Drosophila. IV. Results

of 45 coded compounds tested for the National Toxicology Program Environmental Mutagenesis, 7(3,3), 349-367
Data Type: 1, 4, D sex linked recessive lethal in drosophila

HERO ID: 194373

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 12:

Exposure Route and Method

Medium

X 1

Route is reported as oral dietary study and if no
mutation are induced, the chemical is injected. It
was not reported whether diet was prepared daily to
account for volatility

Domain 4: Test Organism

Metric 13:

Test Animal Characteristics

Medium

x 2

Drosophila stocks and mating schemes were not
reported in text, but cited in (Woodruff et al,
1984; Zimmering et al, 1984; Valencia et al, 1985).
Canton-S males were mated in 3 consecutive harems
with Base females over 7 days

Metric 14:

Adequacy and Consistency of Animal Hus-
bandry Conditions

Low

x 1

Husbandry conditions were not reported.in text.

Metric 15:

Number per Group

High

x 1

At least 20 F2 Base males (or Basc/+ females) were
examined. Statistical analysis (power) was not re-
ported but number is consistent with the study type

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

Not Rated

Testing protocols and experimental methods were
cited in (Woodruff et al, 1984; Zimmering et al,

1984; Valencia et al, 1985).

Metric 17:

Consistency of Outcome Assessment

High

x 1

The outcome assessment was consistent in protocol

and time across all study groups

Metric 18:

Sampling Adequacy

Not Rated

Details regarding sampling adequacy are not appli-
cable for this study type

Metric 19:

Blinding of Assessors

High

x 1

A blind test for induction of SLRLs

Metric 20:

Negative Control Response

High

x 1

The biological response in the negative control group
was adequate

Domain 6: Confounding / Variable Control

Metric 21:

Confounding Variables in Test Design and
Procedures

Low

x 2

Palatability was reported to be part of the dose se-
lection process but is not further described.

Metric 22:

Health Outcomes Unrelated to Exposure

Not Rated

Domain 7: Data Presentation and Analysis

Metric 23:

Statistical Methods

Medium

x 1

Statistical analysis was not conducted, however, suf-
ficient data were provided to allow for other statis-
tical tests..

Metric 24:

Reporting of Data

High

x 2

Quantitative data are reported in table 2 by dose
group and summary data are reported in table 4

Overall Quality Determination

Medium

1.8

Continued on next page . ..

Page 82 of 187

-------
... continued from previous page

Study Citation: J. S. Yoon, J. M. Mason, R. Valencia, R. C. Woodruff, S. Zimmering (1985). Chemical mutagenesis testing in Drosophila. IV. Results

of 45 coded compounds tested for the National Toxicology Program Environmental Mutagenesis, 7(3,3), 349-367
Data Type:	1, 4, D sex linked recessive lethal in drosophila

HERO ID:	194373

Domain

Metric

Rating^

MWF* Score

Comments^

Extracted



Yes







MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; x MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 83 of 187

-------
Table 28: Animal toxicity evaluation results of Kurl et al 1981 for RNA synthesis in rat liver study

Study Citation: R. N. Kurl, L. Poellinger, J. Lund, J. A. Gustafsson (1981). Effects of dioxane on RNA synthesis in the rat liver Archives of Toxicology,

49(1,1), 29-33
Data Type: RNA synthesis in rat liver

HERO ID: 195054

Domain Metric Rating^ MWF* Score Comments^

Domain 1: Test Substance

Metric 1: Test Substance Identity High X 2 2 The test substance was referred to as p-dioxane (1,4-

dioxane).

Metric 2: Test Substance Source High X 1 1 The source of the test substance was reported.

Metric 3: Test Substance Purity Low X 1 3 The purity or grade of the test substance was not

reported.

Domain 2: Test Design

Metric 4: Negative and Vehicle Controls High X 2 2 Concurrent negative controls were utilized (saline in-

jection).

Metric 5: Positive Controls Not Rated NA NA A positive control was not necessary for the end-

point measured in this study, (endogenous RNA
polymerase activity in the liver).

Metric 6: Randomized Allocation Low X 1 3 The animal allocation methodology was not re-

ported.

Domain 3: Exposure Characterization

Metric 7:

Preparation and Storage of Test Substance

Medium

Preparation of the test substance was reported, but
storage of the test substance was not reported (single
dose administered).

Metric 8:

Consistency of Exposure Administration

High

Exposure administration was consistent across treat-
ment groups.

Metric 9:

Reporting of Doses/Concentrations

Medium

Single doses were reported as mg/rat and body
weight was reported as a range (180-200 g).

Metric 10:

Exposure Frequency and Duration

High

The exposure frequency and duration were reported
and appropriate for this endpoint.

Metric 11:

Number of Exposure Groups and Dose Spac-

High

Justification was not provided for the selection of

ing

dose levels; however, the selected doses produced a

range of responses

Metric 12:

Exposure Route and Method

High

The route of exposure was appropriate for this end-

point.

Domain 4: Test Organism

Metric 13: Test Animal Characteristics Low x 2 6 The source of the test animal and health status

were not reported. The age, range of starting body
weights, strain, and sex of the test animal were re-
ported.

Continued on next page ...

Page 84 of 187

-------
. continued from previous page

Study Citation: R. N. Kurl, L. Poellinger, J. Lund, J. A. Gustafsson (1981). Effects of dioxane on RNA synthesis in the rat liver Archives of Toxicology,

49(1,1), 29-33
Data Type: RNA synthesis in rat liver

HERO ID: 195054

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 14:

Adequacy and Consistency of Animal Hus-

Low

X 1

Husbandry conditions were not reported.

bandry Conditions

Metric 15:

Number per Group

High

X 1

The number of animals per treatment group was ad-

equate and appropriate for this endpoint (n = 6).

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

x 2

The outcome assessment methodology was appropri-

ate for this endpoint.

Metric 17:

Consistency of Outcome Assessment

High

x 1

The outcome assessment methodology was consis-

tent across treatment groups.

Metric 18:

Sampling Adequacy

Low

x 1

Number of technical replicates per liver was not re-

ported.

Metric 19:

Blinding of Assessors

Not Rated

This metric is not applicable to the study type.

Metric 20:

Negative Control Response

High

x 1

No response was observed in the negative controls.

Domain 6: Confounding / Variable Control

Metric 21:

Confounding Variables in Test Design and

High

x 2

No differences among starting body weights were re-

Procedures

ported. Food and water consumption were not re-

ported, but this is appropriate for a study of this

type (single dose administered; outcome measured

up to 48 hr later).

Metric 22:

Health Outcomes Unrelated to Exposure

High

x 1

No health outcomes or deaths were reported in the

study.

Domain 7: Data Presentation and Analysis

Metric 23:

Statistical Methods

Unacceptable

x 1

Mean values were reported as mean % of control for

6 rats; however, variance was not given and no sta-

tistical analysis was performed.

Metric 24:

Reporting of Data

High

x 2

Data were reported for all outcomes and all groups.

Overall Quality Determination'

Unacceptable**

1.6

Extracted

MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; X MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

^ This metric met the criteria for high confidence as expected for this type of study

Page 85 of 187

-------
Table 29: Animal toxicity evaluation results for Mcfee et al 1994 for mice bone marrow micronucleus assay

Study Citation: A. F. Mcfee, M. G. Abbott, D. K. Gulati, M. D. Shelby (1994). Results of mouse bone marrow micronucleus studies on 1,4-dioxane

Mutation Research, 322(2,2), 145-148
Data Type: Mouse bone marrow micronucleus assay

HERO ID: 195060

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Test substance is referred to as 1,4-Dioxane and

CASRN is correct.

Metric 2:

Test Substance Source

Low

The source of the test substance is not identified.

Metric 3:

Test Substance Purity

Low

The purity of the test substance is not reported.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

Negative controls were injected with saline.

Metric 5:

Positive Controls

High

Positive controls were injected with mitomycin C
and a positive response was observed.

Metric 6:

Randomized Allocation

Medium

Allocation methods of the study animals were not
reported. However, two laboratories carried out two
trials each, following the same protocol, and it can
be assumed that the results from each location are
sufficiently independent of each other.

Domain 3: Exposure Characterization

Metric 7:

Preparation and Storage of Test Substance

Medium

The preparation of the test substance was briefly re-
ported (dissolved in PBS 2 hr prior to treatment),
but the storage of the test substance was not re-
ported.

Metric 8:

Consistency of Exposure Administration

High

Injection volume and frequency were consistent
across exposure groups.

Metric 9:

Reporting of Doses/Concentrations

High

There was no ambiguity in the administered doses.

Metric 10:

Exposure Frequency and Duration

High

Exposure regimen was appropriate for this endpoint
(daily injections for 1 or 3 days with samples ob-
tained 24-48 hr after last treatment).

Metric 11:

Number of Exposure Groups and Dose Spac-
ing

High

Number of exposure groups and dose spacing was

considered adequate (500, 1000, 2000 mg/kg).

Metric 12:

Exposure Route and Method

High

Intraperitoneal injection is an appropriate route of
administration for the test substance.

Domain 4: Test Organism

Continued on next page . ..

Page 86 of 187

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.. . continued from previous page

Study Citation: A. F. Mcfee, M. G. Abbott, D. K. Gulati, M. D. Shelby (1994). Results of mouse bone marrow micronucleus studies on 1,4-dioxane

Mutation Research, 322(2,2), 145-148
Data Type: Mouse bone marrow micronucleus assay

HERO ID: 195060

Domain Metric Rating^ MWF* Score Comments^

Metric 13:

Test Animal Characteristics

Low

The source of the test animal was not reported. It
is also not clear if the source is identical for the two
laboratories in this study. The starting body weights
were also not reported, although it was included that
all starting body weights were within 4 g of each
other.

Metric 14:

Adequacy and Consistency of Animal Hus-
bandry Conditions

Low

Husbandry conditions were not reported for either
of the two laboratories in this study.

Metric 15:

Number per Group

High

The number of animals per treatment group was ad-
equate and consistent across treatment groups.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

The outcome assessment methodology was appropri-
ate and sensitive.

Metric 17:

Consistency of Outcome Assessment

High

Outcome assessment was consistent across treat-

ment groups and the two laboratories.

Metric 18:

Sampling Adequacy

High

Sampling was adequate. 2,000 polychromatic ery-
throcytes were scored per animal.

Metric 19:

Blinding of Assessors

High

It was reported that slides of bone marrow smears
were coded and two observers scored separate slides
for each animal.

Metric 20:

Negative Control Response

High

Negative control groups yielded negative responses.

Domain 6: Confounding / Variable Control

Metric 21:

Confounding Variables in Test Design and

Medium

Starting body weights were all within 4 g of each

Procedures

other, but the actual values of starting body weights

were not reported. No food or water consumption
data was included, but this is appropriate for this
type of study.

Metric 22:

Health Outcomes Unrelated to Exposure

High

The authors report that no attrition or clinical signs
of toxicity were observed in any treatment group.

Domain 7: Data Presentation and Analysis

Metric 23:

Statistical Methods

Medium

Statistical tests used were appropriate for the data
assuming that data were normally distributed; how-
ever, no test for normality was reported.

Metric 24:

Reporting of Data

High

All data are reported adequately.

Overall Quality Determination1" High 1.5

Extracted Yes

Continued on next page . ..

Page 87 of 187

-------
. continued from previous page

Study Citation: A. F. Mcfee, M. G. Abbott, D. K. Gulati, M. D. Shelby (1994). Results of mouse bone marrow micronucleus studies on 1,4-dioxane

Mutation Research, 322(2,2), 145-148
Data Type:	Mouse bone marrow micronucleus assay

HERO ID:	195060

Domain	Metric	Rating^ MWF* Score	Comments^

* MWF = Metric Weighting Factor

t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

+ The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

if any metric is Unacceptable

Overall rating =

J]. (Metric Score; x MWF;) / J] . MWFj

(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 88 of 187

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Table 30: Animal toxicity evaluation results of Mirkova 1994 for mice bone marrow micronucleus assay

Study Citation: E. T. Mirkova (1994). Activity of the rodent carcinogen 1,4-dioxane in the mouse bone marrow micronucleus assay Mutation Research,

322(2,2), 142-144
Data Type: Mouse bone marrow micronucleus assay

HERO ID: 195062

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

The test substance was identified as 1,4-dioxane with

the correct CASRN.

Metric 2:

Test Substance Source

High

The commercial source of the test substance was re-

ported.

Metric 3:

Test Substance Purity

Medium

The test substance was reported to be "of analytical

grade."

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

Medium

Concurrent negative control groups were included,
but it is not specified whether these animals were
treated with vehicle (water) or left untreated.

Metric 5:

Positive Controls

High

Appropriate concurrent positive control groups were
included (cyclophosphamide oral gavage).

Metric 6:

Randomized Allocation

Low

No random allocation of animals was reported.

Domain 3: Exposure Characterization

Metric 7:

Preparation and Storage of Test Substance

High

Preparation of the test substance was reported.
Storage of the test substance was not reported; how-
ever, the test solutions were prepared immediately
prior to use (single dose administered).

Metric 8:

Consistency of Exposure Administration

High

Exposure administration was reported to be consis-
tent across treatment groups.

Metric 9:

Reporting of Doses/Concentrations

High

Doses were reported without ambiguity.

Metric 10:

Exposure Frequency and Duration

High

The exposure frequency and duration were reported
and appropriate for this endpoint.

Metric 11:

Number of Exposure Groups and Dose Spac-
ing

High

The number of exposure groups and dose spacing
was appropriate.

Metric 12:

Exposure Route and Method

High

The route and method of exposure were appropriate
for the test substance.

Domain 4: Test Organism

Metric 13:

Test Animal Characteristics

Low

The species, strain, and sex of the test animals were
reported. The commercial source, starting body
weight range, and ages were not reported.

Metric 14:

Adequacy and Consistency of Animal Hus-
bandry Conditions

Low

Husbandry conditions were not reported.

Continued on next page . ..

Page 89 of 187

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.. . continued from previous page

Study Citation: E. T. Mirkova (1994). Activity of the rodent carcinogen 1,4-dioxane in the mouse bone marrow micronucleus assay Mutation Research,

322(2,2), 142-144
Data Type: Mouse bone marrow micronucleus assay

HERO ID: 195062

Domain

Metric

Rating^ MWF* Score

Comments^

Metric 15:

Number per Group

High

The number of animals per treatment group was ad-
equate and appropriate for these endpoints (n = 4-
10).

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

The outcome assessment methodology was appropri-
ate for this endpoint.

Metric 17:

Consistency of Outcome Assessment

High

The outcome assessment methodology was consis-
tent across treatment groups.

Metric 18:

Sampling Adequacy

High

Sampling was adequate for the outcome of interest
(2,000 polychromatic erythrocytes per animal).

Metric 19:

Blinding of Assessors

Low

The authors state that slides were prepared and as-

sessed as described previously (Ashby and Mirkova
1987) but do not state specifically that slides were
coded

Metric 20:

Negative Control Response

High

Negative responses were observed in negative con-
trols.

Domain 6: Confounding / Variable Control

Metric 21:

Confounding Variables in Test Design and

Low

Starting body weight ranges were not included.

Procedures

Food and water consumption and respiratory rates

were not reported, but this is appropriate given the
study design.

Metric 22:

Health Outcomes Unrelated to Exposure

High

One of 6 total BALB/c male mice was found dead

at 24 h post-treatment after 5000 mg/kg 1,4-dioxane
administration. This is in line with the 4-day mean
lethal dose (MLD4) identified in a preliminary study,
4500 mg/kg. No other deaths or health outcomes
were reported for any treatment group.

Domain 7: Data Presentation and Analysis

Metric 23:

Statistical Methods

High

The data were analyzed appropriately (one-tailed t-
test) The raw data are provided, enabling an inde-
pendent of the data if necessary.

Metric 24:

Reporting of Data

High

All data were reported adequately.

Overall Quality Determination1"

High

1.5

Extracted

Yes

Continued on next page . ..

Page 90 of 187

-------
... continued from previous page

Study Citation:

E. T. Mirkova (1994). Activity of the rodent carcinogen 1,4-dioxane in the mouse bone

marrow micronucleus assay Mutation Research,



322(2,2), 142-144



Data Type:

Mouse bone marrow micronucleus assay



HERO ID:

195062



Domain

Metric Rating^ MWF* Score

Comments^

MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

4	if any metric is Unacceptable

Overall rating =

J]. (Metric Score; x MWF;) / J] . MWFj

(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 91 of 187

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Table 31: Animal toxicity evaluation results for Miyagawa et al 1999 for DNA synthesis in rat liver study

Study Citation: M. Miyagawa, T. Shirotori, M. Tsuchitani, K. Yoshikawa (1999). Repeat-assessment of 1,4-dioxane in a rat-hepatocyte replicative DNA

synthesis (RDS) test: Evidence for stimulus of hepatocyte proliferation Experimental and Toxicologic Pathology, 51(6,6), 555-558
Data Type: DNA synthesis in rat liver

HERO ID: 195063

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

The test substance was identified as 1,4-dioxane.

Metric 2:

Test Substance Source

High

The source of the test substance was identified.

Metric 3:

Test Substance Purity

Low

Purity of the test substance was not reported.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

Medium

Concurrent negative controls were used, but these

animals were untreated rather than receiving a ve-
hicle (corn oil) gavage.

Metric 5:

Positive Controls

Not Rated

Positive controls were not necessary based on end-

point and study type.

Metric 6:

Randomized Allocation

Low

Animal allocation methodology was not reported.

Domain 3: Exposure Characterization

Metric 7:

Preparation and Storage of Test Substance

High

The test substance preparation was reported, but
the storage of the test substance was not reported
(single-dose administration).

Metric 8:

Consistency of Exposure Administration

High

Exposure administration was consistent across treat-
ment groups.

Metric 9:

Reporting of Doses/Concentrations

High

Doses were reported without ambiguity.

Metric 10:

Exposure Frequency and Duration

High

Exposure frequency and duration were appropriate
for this endpoint, as evidenced by the timecourse
data presented.

Metric 11:

Number of Exposure Groups and Dose Spac-
ing

High

Number of exposure groups and dose spacing were
appropriate and justified for this endpoint (V2, 34,
lx, and 2x the maximum tolerated dose).

Metric 12:

Exposure Route and Method

High

The exposure route and duration were appropriate
for the test substance.

Domain 4: Test Organism

Metric 13:

Test Animal Characteristics

Medium

The test animal starting body weights were not re-
ported. The test animal species, strain, sex, age,
and commercial source were reported.

Metric 14:

Adequacy and Consistency of Animal Hus-
bandry Conditions

Medium

Husbandry conditions were reported to be consistent
across treatment groups, but specific values for tem-
perature, humidity, and light-dark cycle were not
included.

Continued on

next page . .

Page 92 of 187

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.. . continued from previous page

Study Citation: M. Miyagawa, T. Shirotori, M. Tsuchitani, K. Yoshikawa (1999). Repeat-assessment of 1,4-dioxane in a rat-hepatocyte replicative DNA

synthesis (RDS) test: Evidence for stimulus of hepatocyte proliferation Experimental and Toxicologic Pathology, 51(6,6), 555-558
Data Type:	DNA synthesis in rat liver

HERO ID:	195063

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 15:

Number per Group

High

X 1

1

The number of animals per group was appropriate
for these endpoints (n = 3-4).

Domain 5: Outcome Assessment









Metric 16:

Outcome Assessment Methodology

High

x 2

2

The outcome assessment methodologies were appro-
priate for the endpoints of interest.

Metric 17:

Consistency of Outcome Assessment

High

x 1

1

Outcomes were assessed consistently across treat-
ment groups.

Metric 18:

Sampling Adequacy

High

x 1

1

Sampling was adequate for the outcomes of interest
(2,000 hepatocytes per animal).

Metric 19:

Blinding of Assessors

Not Rated

NA

NA

Blinding was not reported; however, lack of blind-
ing is not expected to have a substantial impact on
results for this endpoint ([3H]thymidine or BrdU la-
beling evaluated using fluorescence microscopy).

Metric 20:

Negative Control Response

High

x 1

1

Negative responses were observed in negative control
groups.

Domain 6: Confounding / Variable Control









Metric 21:

Confounding Variables in Test Design and
Procedures

Low

x 2

6

Initial body weights were not reported. Food and
water consumption and respiratory rates were not
reported, but this is appropriate for this study de-
sign.

No attrition or health outcomes were reported in any
treatment group.

Metric 22:

Health Outcomes Unrelated to Exposure

High

x 1

1

Domain 7: Data Presentation and Analysis









Metric 23:

Statistical Methods

Medium

x 1

2

The data were analyzed by t-test, but no test for
normality was reported.

Metric 24:

Reporting of Data

High

x 2

2

All data were adequately reported.

Overall Quality Determination



High



1.5



Extracted



Yes









* MWF = Metric Weighting Factor









' High = 1; Medium = 2; Low =

3; Unacceptable = 4; N/A has no value.









+ The overall rating is calculated

as necessary. EPA may not always provide a comment for a metric that has been categorized as High.



( 4



if any metric is

Unacceptable

Overall rating =

]T\ (Metric Score; X MWF;) / J] . MWFj

(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

U This metric met the criteria for high confidence as expected for this type of study

Page 93 of 187

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Table 32: Animal toxicity evaluation results of Morita and Hayashi 1998 for mouse liver micronucleus assay

Study Citation: T. Morita, M. Hayashi (1998). 1,4-Dioxane is not mutagenic in five in vitro assays and mouse peripheral blood micronucleus assay, but

is in mouse liver micronucleus assay Environmental and Molecular Mutagenesis, 32(3,3), 269-280
Data Type: Mouse liver micronucleus assay

HERO ID: 195065

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

The test substance was identified as 1,4-dioxane with

the correct CASRN.

Metric 2:

Test Substance Source

High

The commercial source of the test substance was re-

ported.

Metric 3:

Test Substance Purity

High

The test substance was reported to be 99.8% pure.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

Appropriate concurrent negative control groups were
included (saline gavage).

Metric 5:

Positive Controls

High

Appropriate concurrent positive control groups were
included (mitomycin C injection).

Metric 6:

Randomized Allocation

High

Random allocation of animals to treatment groups

was reported.

Domain 3: Exposure Characterization

Metric 7:

Preparation and Storage of Test Substance

High

Preparation of the test substance was reported.
Storage of the test substance was not reported; how-
ever, the test solutions were prepared immediately
prior to use (single dose administered).

Metric 8:

Consistency of Exposure Administration

High

Exposure administration was consistent across treat-
ment groups.

Metric 9:

Reporting of Doses/Concentrations

High

Doses were reported without ambiguity.

Metric 10:

Exposure Frequency and Duration

High

The exposure frequency and duration were reported
and appropriate for this endpoint.

Metric 11:

Number of Exposure Groups and Dose Spac-
ing

High

The selected doses were in line with previous oral
gavage studies (listed in Table 1) and produced a
range of responses in the liver micronucleus assay.

Metric 12:

Exposure Route and Method

High

The route and method of exposure were appropriate
for the test substance.

Domain 4: Test Organism

Metric 13:

Test Animal Characteristics

High

The species, strain, age, sex, starting body weight
range, and commercial source was provided for the
test animals.

Continued on next page . ..

Page 94 of 187

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.. . continued from previous page

Study Citation: T. Morita, M. Hayashi (1998). 1,4-Dioxane is not mutagenic in five in vitro assays and mouse peripheral blood micronucleus assay, but

is in mouse liver micronucleus assay Environmental and Molecular Mutagenesis, 32(3,3), 269-280
Data Type: Mouse liver micronucleus assay

HERO ID: 195065

Domain

Metric

Rating^ MWF* Score

Comments^

Metric 14:

Adequacy and Consistency of Animal Hus-

Medium

Husbandry conditions were reported to be consistent

bandry Conditions

across treatment groups, but specific values for tem-

perature, humidity, and light-dark cycle were not
included.

Metric 15:

Number per Group

High

The number of animals per treatment group was ade-
quate and appropriate for these endpoints (n = 4-5).

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

The outcome assessment methodology was appropri-
ate for this endpoint.

Metric 17:

Consistency of Outcome Assessment

High

The outcome assessment methodology was consis-
tent across treatment groups.

Metric 18:

Sampling Adequacy

High

Sampling was adequate for the outcomes of interest
(2,000 hepatocytes per animal).

Metric 19:

Blinding of Assessors

Low

Authors state that selection and scoring were accord-

ing to published criteria Braithwaithe and Ashby
1988 (in which slides are coded) but the authors do
not specifically state whether slides were coded in
this study

Metric 20:

Negative Control Response

High

Negative responses were observed in negative con-
trols.

Domain 6: Confounding / Variable Control

Metric 21:

Confounding Variables in Test Design and

High

A range for initial body weights was reported. Food

Procedures

and water consumption and respiratory rates were

not reported, but this is appropriate given the study
design.

Metric 22:

Health Outcomes Unrelated to Exposure

High

No attrition or health outcomes were reported in any
treatment group.

Domain 7: Data Presentation and Analysis

Metric 23:

Statistical Methods

High

Kastenbaum & Bowman's table was used to compare

percent micronucleus results. In addition, individual
animal data are provided, enabling re-analysis using
different statistical procedures if necessary.

Metric 24:

Reporting of Data

High

All data were reported adequately.

Overall Quality Determination1"

High

1.1

Extracted

Yes

Continued on next page . ..

Page 95 of 187

-------
. continued from previous page

Study Citation: T. Morita, M. Hayashi (1998). 1,4-Dioxane is not mutagenic in five in vitro assays and mouse peripheral blood micronucleus assay, but

is in mouse liver micronucleus assay Environmental and Molecular Mutagenesis, 32(3,3), 269-280
Data Type:	Mouse liver micronucleus assay

HERO ID:	195065

Domain	Metric	Rating^ MWF* Score	Comments^

* MWF = Metric Weighting Factor

t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

+ The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

if any metric is Unacceptable

Overall rating =

J]. (Metric Score; x MWF;) / J] . MWFj

(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 96 of 187

-------
Table 33: Animal toxicity evaluation results for Tinwell and Ashby 1994 for bone marrow micronucleus assay in mice

Study Citation: H. Tinwell, J. Ashby (1994). Activity of 1,4-dioxane in mouse bone marrow micronucleus assays Mutation Research, 322(2,2), 148-150
Data Type: Bone Marrow Micronucleus assay in Mouse

HERO ID: 195086

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

The test substance was identified as 1,4-dioxane.

Metric 2:

Test Substance Source

High

The source of the test substance was identified. The

product number and batch/lot number were not re-
ported; however the material is not expected to vary
in composition.

Metric 3:

Test Substance Purity

Low

The purity of the test substance was not reported.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

Concurrent negative controls were used; dosed with
vehicle (distilled water)

Metric 5:

Positive Controls

High

An appropriate concurrent positive control was used.

Metric 6:

Randomized Allocation

Low

Animal allocation methodology was not reported.

Domain 3: Exposure Characterization

Metric 7:

Preparation and Storage of Test Substance

Medium

The test substance preparation was described. The
storage of the test substance was not reported; how-
ever, omission of these details are unlikely to have a
substantial impact on the results (single dose study).

Metric 8:

Consistency of Exposure Administration

High

Exposure administration was consistent across treat-
ment groups.

Metric 9:

Reporting of Doses/Concentrations

High

Doses were reported without ambiguity.

Metric 10:

Exposure Frequency and Duration

High

Exposure frequency and duration were appropriate
for this endpoint; single oral dose

Metric 11:

Number of Exposure Groups and Dose Spac-
ing

High

There was one exposure group per experiment, with
3 experiments; the administered dose levels were jus-
tified.

Metric 12:

Exposure Route and Method

High

The exposure route was appropriate for the test sub-
stance

Domain 4: Test Organism

Metric 13:

Test Animal Characteristics

Medium

The test animal species, strain, and sex were re-
ported (health status, and starting body weight were
not reported). The commercial source or in-house
colony was not specified; however, these details were
noted to have been described previously in a related
study (Ashby and Mirkova, 1987). The test species

and strain were an appropriate animal model for the
evaluation of this endpoint.

Continued on next page . ..

Page 97 of 187

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. continued from previous page

Study Citation: H. Tinwell, J. Ashby (1994). Activity of 1,4-dioxane in mouse bone marrow micronucleus assays Mutation Research, 322(2,2), 148-150
Data Type: Bone Marrow Micronucleus assay in Mouse

HERO ID: 195086

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 14:

Adequacy and Consistency of Animal Hus-
bandry Conditions

Low

Husbandry conditions were not reported; however,
these details were noted to have been described pre-
viously in a related study (Ashby and Mirkova, 1987)

Metric 15:

Number per Group

High

The number of animals per group was appropriate
for the study type and endpoints (n = 3-8).

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

The outcome assessment methodologies were appro-
priate for the endpoints of interest.

Metric 17:

Consistency of Outcome Assessment

Medium

There were minor differences in the outcome assess-
ment protocol, but these uncertainties or limitations
are unlikely to have substantial impact on results.
The studies were performed during a transition from
the use of the Giemsa stain to acridine orange stain
for evaluating bone marrow smears.

Metric 18:

Sampling Adequacy

High

Sampling was adequate for the outcomes of interest
(2,000 polychromatic erythrocytes per animal).

Metric 19:

Blinding of Assessors

Low

Blinding was not specifically reported in this study;
The authors state that slides were prepared as de-
scribed previously (Tinwell and Ashby, 1989), but
blinding/coding of slides is not described in that pa-
per either.

Metric 20:

Negative Control Response

Low

The biological responses of the negative control
groups were reported; however, there were deficien-
cies regarding the control responses that may have a
substantial impact on results. One control animal in
experiment 3 had an elevated MPE frequency which
affected the determination of biological significance
of treated mice.

Domain 6: Confounding / Variable Control

Metric 21:

Confounding Variables in Test Design and
Procedures

Medium

Initial body weights were not reported. Food and
water consumption were not reported, but this is
appropriate for this study design.

Metric 22:

Health Outcomes Unrelated to Exposure

Medium

Data on attrition and/or health outcomes unrelated
to exposure for each study group were not reported.

Domain 7: Data Presentation and Analysis

Metric 23:

Statistical Methods

High

Statistical methods were clearly described and ap-

propriate for the dataset (one sided students t-test).

Metric 24:

Reporting of Data

High

All data were adequately reported.

Overall Quality Determination'

High

1.5

Extracted

Yes

Continued on next page . ..

Page 98 of 187

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. continued from previous page

Study Citation: H. Tinwell, J. Ashby (1994). Activity of 1,4-dioxane in mouse bone marrow micronucleus assays Mutation Research, 322(2,2), 148-150
Data Type:	Bone Marrow Micronucleus assay in Mouse

HERO ID:	195086

Domain	Metric	Rating^ MWF* Score	Comments^

* MWF = Metric Weighting Factor

t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

+ The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

if any metric is Unacceptable

Overall rating =

J]. (Metric Score; x MWF;) / J] . MWFj

(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating,
ft This metric met the criteria for high confidence as expected for this type of study

Page 99 of 187

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Table 34: Animal toxicity evaluation results of Morita 1994 for mouse peripheral blood micronucleus assay

Study Citation: T. Morita (1994). No clastogenicity of 1,4 dioxane as examined in the mouse peripheral blood micronucleus test Honyu Dobutsu Shiken

Bunkakai Kaiho, 2 7-8
Data Type: Mouse peripheral blood micronucleus assay

HERO ID: 196085

Domain

Metric

Rating^ MWF* Score

Comments^

Domain 1: Test Substance

Metric

Test

Metric

Test

Metric

Test

High
High

x 2
x 1

Medium x 1

2 The test substance was identified as 1,4-dioxane with

the correct CASRN.

1 The commercial source and lot number of the test
substance was reported.

2 The purity of the test substance was not reported;
however, the commercial source and lot number were
identified, making it potentially possible to obtain
the purity of that lot. This is not expected to have
adversely affected the results.

Domain 2: Test Design

Metric 4: Negative and Vehicle Controls

Metric 5: Positive Controls

Metric 6: Randomized Allocation

High X 2 2 Appropriate concurrent negative control groups were

included (saline).

High X 1 1 Appropriate concurrent positive control groups were

included (mitomycin C) and a positive result was
observed.

High X 1 1 Random allocation of animals to treatment groups

was reported.

Domain 3: Exposure Characterization

Metric

Preparation and Storage of Test Substance

High

Preparation of the test substance was reported.
Storage of the test substance was not reported; how-
ever, the test solutions were prepared immediately
prior to use (single dose administered).

Metric

Consistency of Exposure Administration

High

Exposure administration was consistent across treat-
ment groups.

Metric

Reporting of Doses/Concentrations

High

Doses were reported without ambiguity.

Metric

10:

Exposure Frequency and Duration

High

The exposure frequency and duration were reported
and appropriate for this endpoint.

Metric

11:

Number of Exposure Groups and Dose Spac-

High

The selected doses were based off the maximum tol-

ing

erated dose (3200 mg/kg), determined in a prelim-

inary study. The number of exposure groups and
dose spacing were reported and appropriate.

Metric

12:

Exposure Route and Method

High

The route and method of exposure were appropriate
for the test substance.

Domain 4: Test Organism

Continued on next page

Page 100 of 187

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.. . continued from previous page

Study Citation: T. Morita (1994). No clastogenicity of 1,4 dioxane as examined in the mouse peripheral blood micronucleus test Honyu Dobutsu Shiken

Bunkakai Kaiho, 2 7-8
Data Type: Mouse peripheral blood micronucleus assay

HERO ID: 196085

Domain Metric Rating^ MWF* Score Comments^

Metric 13:

Test Animal Characteristics

Medium

The species, strain, age, sex, starting body weight
range, and commercial source was provided for the
test animals. Health status was not reported

Metric 14:

Adequacy and Consistency of Animal Hus-
bandry Conditions

Low

Husbandry conditions were not reported.

Metric 15:

Number per Group

High

The number of animals per treatment group was ad-
equate and appropriate for these endpoints (n = 5).

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

The outcome assessment methodology was appropri-
ate for this endpoint.

Metric 17:

Consistency of Outcome Assessment

High

The outcome assessment methodology was consis-
tent across treatment groups.

Metric 18:

Sampling Adequacy

High

Sampling was adequate for the outcomes of interest
(1,000 reticulocytes assessed per animal per time-
point).

Metric 19:

Blinding of Assessors

Low

blinding of assessors was not reported

Metric 20:

Negative Control Response

High

Negative responses were observed in negative con-
trols.

Domain 6: Confounding / Variable Control

Metric 21:

Confounding Variables in Test Design and

High

A range for initial body weights was reported. Food

Procedures

and water consumption and respiratory rates were

not reported, but this is appropriate given the study
design.

Metric 22:

Health Outcomes Unrelated to Exposure

High

One mouse in the highest dose group died between
48 and 72 hr post-treatment. This is considered to
be treatment-related, as the highest dose was se-
lected based on the maximum tolerated dose, deter-
mined in a preliminary study. No attrition occurred
in any other treatment group, and no adverse health
outcomes or clinical signs of toxicity were reported.

Domain 7: Data Presentation and Analysis

Metric 23:

Statistical Methods

High

Kastenbaum & Bowman's table was used to compare

percent micronucleus results. In addition, individ-
ual animal data are provided, enabling a potential
reanalysis using a different statistical test.

Metric 24:

Reporting of Data

High

All data were reported adequately.

Overall Quality Determination"1' High ~L2

Extracted Yes

Continued on next page . ..

Page 101 of 187

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... continued from previous page

Study Citation: T. Morita (1994). No clastogenicity of 1,4 dioxane as examined in the mouse peripheral blood micronucleus test Honyu Dobutsu Shiken

Bunkakai Kaiho, 2 7-8
Data Type:	Mouse peripheral blood micronucleus assay

HERO ID:	196085

Domain

Metric

Rating^ MWF* Score

Comments^



MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; x MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 102 of 187

-------
Table 35: Animal toxicity evaluation results of Stott et al 1981 for in vivo DNA synthesis, alkylation and repair in rats

Study Citation: W. T. Stott, J. F. Quast, P. G. Watanabe (1981). Differentiation of the mechanisms of oncogenicity of 1,4-dioxane and 1,3-

hexachlorobutadiene in the rat Toxicology and Applied Pharmacology, 60(2,2), 287-300
Data Type: In vivo DNA synthesis, alkylation and repair

HERO ID: 1937837

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Identified by established nomenclature as 1,4-

dioxane.

Metric 2:

Test Substance Source

High

Commercial source of radiolabeled and unlabeled

1,4-dioxane was reported.

Metric 3:

Test Substance Purity

High

Purity was >99% for unlabeled compound; radio-

chemical purity was >98%.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

Negative controls were used for the DNA synthe-

sis experiments (saline for acute gavage exposure;

drinking water for repeated dose exposure); negative

controls were not needed for the DNA alkylation or

repair experiments.

Metric 5:

Positive Controls

High

Dimethylnitrosamine was used as a positive control

for the DNA alkylation and repair experiments and

positive responses were observed.

Metric 6:

Randomized Allocation

High

Computer randomization was used to asign animals

to study groups.

Domain 3: Exposure Characterization

Metric 7:

Preparation and Storage of Test Substance

Medium

Test solutions were prepared in saline. Storage was

not described; however DNA alkylation and repair

assays (and acute DNA synthesis assays) were single

dose experiments, suggesting that omission of these

details are unlikely to have a substantial impact on

the results.

Metric 8:

Consistency of Exposure Administration

High

Exposures were administered consistently across

study groups.

Metric 9:

Reporting of Doses/Concentrations

Medium

Nominal concentrations for gavage exposures were

reported; Nominal concentration administered in

drinking water was reported, but actual doses were

not reported; water intake rates and body weights

were not reported

Metric 10:

Exposure Frequency and Duration

High

Frequency and duration were appropriate for this

study type and outcome(s) of interest.

Continued on

next page .

Page 103 of 187

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.. . continued from previous page

Study Citation: W. T. Stott, J. F. Quast, P. G. Watanabe (1981). Differentiation of the mechanisms of oncogenicity of 1,4-dioxane and 1,3-

hexachlorobutadiene in the rat Toxicology and Applied Pharmacology, 60(2,2), 287-300
Data Type: In vivo DNA synthesis, alkylation and repair

HERO ID: 1937837

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 11:

Number of Exposure Groups and Dose Spac-

Medium

Doses were selected based on previous carcinogenic-

ing

ity studies (i.e., tumorigenic and non tumorigenic

doses). 3 doses were used for acute studies of DNA
synthesis; however only two doses were used for re-
peated dose exposures and a single high dose was
used for DNA alkylation and repair assays.

Metric 12:

Exposure Route and Method

High

Oral gavage/drinking water administration is appro-
priate for the test substance.

Domain 4: Test Organism

Metric 13:

Test Animal Characteristics

Medium

The test animal species, strain, sex, and starting
body weight were reported (age and health status
were not reported). Animals were obtained from a
commercial laboratory.

Metric 14:

Adequacy and Consistency of Animal Hus-

Low

The study reports that rats were housed in "envi-

bandry Conditions

ronmentally controlled animal holding rooms" but

details of husbandry conditions were not sufficiently
reported.

Metric 15:

Number per Group

Medium

The number of animals per study group was reported
and was low (4-6/group) for the DNA synthesis and
repair assays. Only 2 animals were used to evaluate
DNA alkylation.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

Medium

Outcome assessment methods were well described

and appropriate and sensitive for the outcomes of
interest; scintillation counting methodology for eval-
uating DNA repair is relatively insensitive.

Metric 17:

Consistency of Outcome Assessment

High

Outcomes were assessed consistently across study
groups.

Metric 18:

Sampling Adequacy

High

Sampling for the outcome of interest was reported
and adequate

Metric 19:

Blinding of Assessors

Not Rated

This metric is not applicable for the outcomes of

interest (no subjective outcomes).

Metric 20:

Negative Control Response

High

Negative control response was reported for DNA
content and DNA synthesis following acute and re-
peated dose studies; relevant positive and negative
control responses were reported for DNA repair.

Domain 6: Confounding / Variable Control

Metric 21: Confounding Variables in Test Design and Low x 2 6 Initial body weight and food/water intake were not

Procedures reported for each study group. These deficiencies are

likely to affect the results of the repeat dose DNA
synthesis assay (11 week drinking water exposure).

Continued on next page . ..

Page 104 of 187

-------
. continued from previous page

Study Citation: W. T. Stott, J. F. Quast, P. G. Watanabe (1981). Differentiation of the mechanisms of oncogenicity of 1,4-dioxane and 1,3-

hexachlorobutadiene in the rat Toxicology and Applied Pharmacology, 60(2,2), 287-300
Data Type:	In vivo DNA synthesis, alkylation and repair

HERO ID:	1937837

Domain Metric

Rating^

MWF*

Score

Comments^

Metric 22: Health Outcomes Unrelated to Exposure

Low

X 1

3

Data on attrition and/or health outcomes unrelated
to exposure were not reported for each study group

Domain 7: Data Presentation and Analysis

Metric 23: Statistical Methods

Metric 24: Reporting of Data

High
High

X 1

x 2

1

2

Statistical methods were clearly described and ap-
propriate for in vivo dataset(s) (Dunnett's t test).

Data for exposure-related findings were presented
for all in vivo outcomes by exposure group (mean
+ /1 SD).

Overall Quality Determination1"

High



1.6



Extracted

Yes









MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; x MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 105 of 187

-------
Table 36: Animal toxicity evaluation results of Fujioka et al 2019 for in vivo mutations in rats

Study Citation: M. Gi, M. Fujioka, A. Kakehashi, T. Okuno, K. Masumura, T. Nohmi, M. Matsumoto, M. Omori, H. Wanibuchi, S. Fukushima
(2018). In vivo positive mutagenicity of 1,4-dioxane and quantitative analysis of its mutagenicity and carcinogenicity in rats Archives
of Toxicology, 92(10,10), 3207-3221
Data Type: 16-week drinking water study in F344 rats) in vivo mutation assay

HERO ID: 5029473

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

The test substance was identified as 1,4-dioxane.

Metric 2:

Test Substance Source

High

The source of the test substance was identified. The

product number and batch/lot number was not re-
ported; however the material is not expected to vary
in composition.

Metric 3:

Test Substance Purity

High

The test substance purity was reported (> 99.9%)

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

Concurrent negative controls were tested (untreated
drinking water) for all 3 experiments.

Metric 5:

Positive Controls

Not Rated

The use of positive controls was not applicable for

this study type.

Metric 6:

Randomized Allocation

Low

The study did not report how animals were allocated
to study groups for any of the three experiments.

Domain 3: Exposure Characterization

Metric 7:

Preparation and Storage of Test Substance

Low

The test substance was dissolved in drinking water
(not further described). Storage of the test sub-
stance was not reported and exposure was for 16
weeks.

Metric 8:

Consistency of Exposure Administration

High

Exposure administration was consistent across treat-
ment groups in all three experiments.

Metric 9:

Reporting of Doses/Concentrations

High

Concentration were reported without ambiguity.
Concentrations reported in ppm drinking water.
Measured water intake and 1,4-dioxane intake was
reported.

Metric 10:

Exposure Frequency and Duration

High

Exposure frequency and duration were appropriate
for this endpoint

Metric 11:

Number of Exposure Groups and Dose Spac-

Medium

Though the study authors did not justify the num-

ing

ber of exposure groups or concentration spacing, the

number of exposure groups and spacing of exposure
levels appear to be adequate to show results relevant
to the outcome of interest.

Metric 12:

Exposure Route and Method

High

The exposure route was appropriate for the test sub-
stance.

Continued on next page . ..

Page 106 of 187

-------
.. . continued from previous page

HERO ID: 5029473

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 4: Test Organism

Metric 13:

Test Animal Characteristics

Medium

The test animal species, strain, sex, and age were re-
ported while health status and starting body weight
were not. It was noted that body weight was mea-
sured weekly. The test animal was from a reported
commercial source. The test species and strain were
an appropriate animal model for the evaluation of
this endpoint. The uncertainties in reporting are
unlikely to have a substantial impact on results.

Metric 14:

Adequacy and Consistency of Animal Hus-
bandry Conditions

High

All husbandry conditions were reported and were ad-
equate.

Metric 15:

Number per Group

Medium

The number of animals per study group was re-
ported; while slightly lower than typical for sub-
chronic studies for some endpoints (N=5-8), it was
sufficient for statistical analysis.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

The outcome assessment methodologies were appro-
priate for the endpoints of interest.

Metric 17:

Consistency of Outcome Assessment

High

The outcome assessment was carried out consis-

tently for all three experiments.

Metric 18:

Sampling Adequacy

High

Sampling was adequate for the outcomes of interest

Metric 19:

Blinding of Assessors

Not Rated

Automated procedures; details referenced in another

publication.

Metric 20:

Negative Control Response

High

The biological response of the negative control
groups were adequate

Domain 6: Confounding / Variable Control

Metric 21:

Confounding Variables in Test Design and

Medium

Initial body weights were not reported; though

Procedures

drinking water and food consumption was reported.

These minor uncertainties are unlikely to have a sub-
stantial impact on results. There were no other con-
founding variables noted in the study.

Metric 22:

Health Outcomes Unrelated to Exposure

Medium

Data on attrition and/or health outcomes unrelated
to exposure for each study group were not reported.

Domain 7: Data Presentation and Analysis

Metric 23:

Statistical Methods

High

Statistical methods were clearly described and ap-

propriate for the dataset.

Metric 24:

Reporting of Data

High

All data were adequately reported.

Continued on next page . ..

Page 107 of 187

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.. . continued from previous page

Study Citation:

Data Type:
HERO ID:

M. Gi, M. Fujioka, A. Kakehashi, T. Okuno, K. Masumura, T. Nohmi, M. Matsumoto, M. Omori, H. Wanibuchi, S. Fukushima
(2018). In vivo positive mutagenicity of 1,4-dioxane and quantitative analysis of its mutagenicity and carcinogenicity in rats Archives
of Toxicology, 92(10,10), 3207-3221

16-week drinking water study in F344 rats) in vivo mutation assay
5029473

Domain

Metric

Rating^

MWF* Score

Comments^

Overall Quality Determination1"



High

1.4



Extracted



Yes







MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; X MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

This metric met the criteria for high confidence as expected for this type of study

Page 108 of 187

-------
Table 37: In vitro evaluation results of Sina 1983 for mutagenesis in rat hepatocyte assay

Study Citation: J. F. Sina, C. L. Bean, G. R. Dysart, V. I. Taylor, M. 0. Bradley (1983). Evaluation of the alkaline elution/rat hepatocyte assay as a
predictor of carcinogenic/mutagenic potential Mutation Research: Environmental Mutagenesis and Related Subjects, 113(5,5), 357-391
Data Type: DNA damage (SSB) in rat hepatocytes for 1,4-dioxane

HERO ID: 7323

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

The test substance was identified as 1,4-dioxane with

the correct CASRN.

Metric 2:

Test Substance Source

Medium

The commercial source of the test substance was
identified, but lot number was not reported.

Metric 3:

Test Substance Purity

Low

Purity of the test substance was not identified.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

Medium

Negative controls were included. It was not speci-

fied whether the negative controls were treated with
water, DMSO, or left untreated.

Metric 5:

Positive Controls

High

Dimethylnitrosamine was utilized as a positive con-
trol in each assay. Positive results were obtained
from positive control groups. This compound re-
quires metabolic activation and was also utilized as
a validation of hepatocyte metabolism.

Metric 6:

Assay Procedures

High

Assay procedures were well-described.

Metric 7:

Standards for Tests

High

The QC criteria were adequate to demonstrate va-

lidity, acceptability, and reliability of this test.

Domain 3: Exposure Characterization

Metric 8:

Preparation and Storage of Test Substance

High

The preparation of the test substance was reported.
The storage of the test substance was not reported
(single dose administration).

Metric 9:

Consistency of Exposure Administration

High

Exposure administration was consistent across treat-
ment groups.

Metric 10:

Reporting of Doses/Concentrations

High

Exposure concentrations were reported without am-
biguity.

Metric 11:

Number of Exposure Groups and Concentra-
tion Spacing

High

The exposure duration (3 hr) was reported and ap-
propriate for the outcome of interest.

Metric 12:

Exposure Route and Method

High

Number of exposure groups and dose spacing was
reported and appropriate.

Metric 13:

Metabolic Activation

High

This assay did not include an exogenous metabolic

activation step, as the cells used were primary rat
hepatocytes.

Domain 4: Test Model

Continued on next page . ..

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.. . continued from previous page

Study Citation: J. F. Sina, C. L. Bean, G. R. Dysart, V. I. Taylor, M. O. Bradley (1983). Evaluation of the alkaline elution/rat hepatocyte assay as a
predictor of carcinogenic/mutagenic potential Mutation Research: Environmental Mutagenesis and Related Subjects, 113(5,5), 357-391
Data Type: DNA damage (SSB) in rat hepatocytes for 1,4-dioxane

HERO ID: 7323

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 14:

Test Model

Medium

x 2

The identity and origin of the test model were re-
ported. No additional information was provided.

Metric 15:

Number per Group

Low

x 1

The number of plates independently treated with
1,4-dioxane is not specified (although 2 repli-
cates/plate was indicated). This may suggest the
use of a single culture per concentration. .

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

x 2

The outcome assessment methodology was appropri-
ate for the intended outcome of interest.

Metric 17:

Consistency of Outcome Assessment

High

x 1

The outcome assessment methodology was consis-
tent across treatment groups.

Metric 18:

Sampling Adequacy

Not Rated

This metric is not applicable to the outcome.

Metric 19:

Blinding of Assessors

Not Rated

This metric is not applicable to the study type.

Domain 6: Confounding / Variable Control

Metric 20:

Confounding Variables in Test Design and
Procedures

High

x 2

There were no differences reported in protocols
across treatment groups.

Metric 21:

Confounding Variables in Outcomes Unre-
lated to Exposure

High

x 1

No confounding variables were reported.

Domain 7: Data Presentation and Analysis

Metric 22:

Data Analysis

High

x 1

Statistical analysis was not conducted. A three-fold

increase in DNA single-strand breaks over negative
controls was considered to be a positive result. Raw
data are available for statistical analysis.

Metric 23:

Data Interpretation

High

x 2

The evaluation criteria (DNA single-strand breaks)
are consistent with current standards.

Metric 24:

Cytotoxicity Data

High

x 1

The cytotoxicity of 1,4-dioxane was measured by try-
pan blue dye exclusion for all doses and by release of
glutamate-oxaloacetate transaminase (GOT) from
the cells at the two lowest doses. The methods were
adequately described for each cytotoxicity assay.

Metric 25:

Reporting of Data

High

x 2

Data were reported adequately.

Overall Quality Determination

High

1.3

Extracted

Yes

Continued on next page . ..

Page 110 of 187

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... continued from previous page

Study Citation: J. F. Sina, C. L. Bean, G. R. Dysart, V. I. Taylor, M. O. Bradley (1983). Evaluation of the alkaline elution/rat hepatocyte assay as a
predictor of carcinogenic/mutagenic potential Mutation Research: Environmental Mutagenesis and Related Subjects, 113(5,5), 357-391
Data Type:	DNA damage (SSB) in rat hepatocytes for 1,4-dioxane

HERO ID:	7323

Domain	Metric	Rating^ MWF* Score	Comments^

* MWF = Metric Weighting Factor

t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

+ The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

if any metric is Unacceptable

Overall rating =

J]. (Metric Score; x MWF;) / J] . MWFj

(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 111 of 187

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Table 38: In vitro evaluation results of Galloway et al 1987 for chromosomal aberration study in Chinese hamster ovary cells

Study Citation: S. M. Galloway, M. J. Armstrong, C. Reuben, S. Colman, B. Brown, C. Cannon, A. D. Bloom, F. Nakamura, M. Ahmed, S. Duk, J.

Rimpo, B. H. Margolin, M. A. Resnick, B. Anderson, E. Zeiger (1987). Chromosome aberrations and sister chromatid exchanges in
Chinese hamster ovary cells: evaluations of 108 chemicals Environmental and Molecular Mutagenesis, 10(Suppl. 10,Suppl. 10), 1-175
Data Type: 1,4-Dioxane in vitro chromosomal aberration

HERO ID: 7768

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

The test substances were identified using established

nomenclature and CASRN.

Metric 2:

Test Substance Source

High

The test substances were obtained from Litton Bio-

netics, Inc.

Metric 3:

Test Substance Purity

Low

Purity of the test substances were not reported.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

Solvent controls were employed appropriately

Metric 5:

Positive Controls

High

Two positive controls were employed (triethylen-

emelamine or mitomycin C and cyclophosphamide);

their response was appropriate (significant increase

in chromosomal aberrations).

Metric 6:

Assay Procedures

High

Assay procedures were well described.

Metric 7:

Standards for Tests

Not Rated

Not applicable to this study design.

Domain 3: Exposure Characterization

Metric 8:

Preparation and Storage of Test Substance

Medium

General information regarding test substance prepa-

ration was included (e.g., dissolving in solvent imme-

diately before use), but storage conditions were not

provided.

Metric 9:

Consistency of Exposure Administration

High

Information regarding exposure administration was

reported and consistency of administration across

groups is inferred from the text.

Metric 10:

Reporting of Doses/Concentrations

High

Exposure doses were reported for each trial.

Metric 11:

Number of Exposure Groups and Concentra-

High

Exposure duration was clearly stated and appropri-

tion Spacing

ate for the endpoint.

Metric 12:

Exposure Route and Method

High

Dose selection was described in detail and based on

preliminary growth inhibition tests, followed by ob-

servations of cell monolayer confluence and mitotic

activity to maximize available metaphase cells. The

number of exposure groups was consistent for the

Metric 13:

Metabolic Activation

High

Tests were run with and without metabolic activa-

tion. Preparation of S9 mix was described in detail.

Domain 4: Test Model

Continued on next page . ..

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.. . continued from previous page

Study Citation: S. M. Galloway, M. J. Armstrong, C. Reuben, S. Colman, B. Brown, C. Cannon, A. D. Bloom, F. Nakamura, M. Ahmed, S. Duk, J.

HERO ID: 7768

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 14:

Test Model

High

Test models were described in detail and appropriate

for the endpoints assessed.

Metric 15:

Number per Group

Low

There was only one study group for each of the three
exposure concentrations tests (i.e., no replicates).

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

The assessment methodology addressed the intended
outcomes of interest.

Metric 17:

Consistency of Outcome Assessment

High

Outcome assessment protocol was consistent across
study groups.

Metric 18:

Sampling Adequacy

Medium

The number of cells/dose (100) was reported and is
slightly less than appropriate.

Metric 19:

Blinding of Assessors

High

Test substance was supplied under code; assessors
did not know its identity until after scoring; slides
were coded for scoring.

Domain 6: Confounding / Variable Control

Metric 20:

Confounding Variables in Test Design and
Procedures

High

There were no confounding variables in test design
or procedures that were reported by study authors.

Metric 21:

Confounding Variables in Outcomes Unre-
lated to Exposure

High

There were no confounding variables reported unre-
lated to exposure.

Domain 7: Data Presentation and Analysis

Metric 22:

Data Analysis

High

Statistical analyses were clearly described and pre-
sented in results tables.

Metric 23:

Data Interpretation

High

Data were reported in such a way as to allow inter-
pretation of test results.

Metric 24:

Cytotoxicity Data

Medium

Cytotoxicity endpoints such as induction of cell
death and delay in cell cycle progression were noted,
and selected exposure doses were based on relation
to toxicity. However, methods of measurement for
specific cytotoxicity endpoints were not described.

Metric 25:

Reporting of Data

High

Data were presented for percent cells with aberra-
tions in three ways for each exposure concentration:
total, simple, and complex aberrations.

Overall Quality Determination

High

1.2

Extracted

Yes

Continued on next page . ..

Page 113 of 187

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.. . continued from previous page

Study Citation:

Data Type:
HERO ID:

S. M. Galloway, M. J. Armstrong, C. Reuben, S. Colman, B. Brown, C. Cannon, A. D. Bloom, F. Nakamura, M. Ahmed, S. Duk, J.
Rimpo, B. H. Margolin, M. A. Resnick, B. Anderson, E. Zeiger (1987). Chromosome aberrations and sister chromatid exchanges in
Chinese hamster ovary cells: evaluations of 108 chemicals Environmental and Molecular Mutagenesis, 10(Suppl. 10,Suppl. 10), 1-175
1,4-Dioxane in vitro chromosomal aberration
7768

Domain

Metric

Rating^ MWF* Score

Comments^

MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; X MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

This metric met the criteria for high confidence as expected for this type of study

Page 114 of 187

-------
Table 39: In vitro evaluation results of Galloway et al 1987 for sister chromatid exchanges study in Chinese hamster ovary cells

Study Citation: S. M. Galloway, M. J. Armstrong, C. Reuben, S. Colman, B. Brown, C. Cannon, A. D. Bloom, F. Nakamura, M. Ahmed, S. Duk, J.

HERO ID: 7768

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

The test substances were identified using established

nomenclature and CASRN.

Metric 2:

Test Substance Source

High

The test substances were obtained from Litton Bio-

netics, Inc.

Metric 3:

Test Substance Purity

Low

Purity of the test substances were not reported.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

Solvent controls were employed appropriately

Metric 5:

Positive Controls

High

Two positive controls were employed (triethylen-

emelamine or mitomycin C and cyclophosphamide);

their response was appropriate (significant increase

in chromosomal aberrations).

Metric 6:

Assay Procedures

High

Assay procedures were well described.

Metric 7:

Standards for Tests

Not Rated

Not applicable to this study design.

Domain 3: Exposure Characterization

Metric 8:

Preparation and Storage of Test Substance

Medium

General information regarding test substance prepa-

ration was included (e.g., dissolving in solvent imme-

diately before use), but storage conditions were not

provided.

Metric 9:

Consistency of Exposure Administration

High

Information regarding exposure administration was

reported and consistency of administration across

groups is inferred from the text.

Metric 10:

Reporting of Doses/Concentrations

High

Exposure doses were reported for each trial.

Metric 11:

Number of Exposure Groups and Concentra-

High

Exposure duration was clearly stated and appropri-

tion Spacing

ate for the endpoint.

Metric 12:

Exposure Route and Method

High

Dose selection was described in detail and based on

preliminary growth inhibition tests, followed by ob-

servations of cell monolayer confluence and mitotic

activity to maximize available metaphase cells. The

number of exposure groups was consistent for the

Metric 13:

Metabolic Activation

High

Tests were run with and without metabolic activa-

tion. Preparation of S9 mix was described in detail.

Domain 4: Test Model

Continued on next page . ..

Page 115 of 187

-------
. continued from previous page

Study Citation: S. M. Galloway, M. J. Armstrong, C. Reuben, S. Colman, B. Brown, C. Cannon, A. D. Bloom, F. Nakamura, M. Ahmed, S. Duk, J.

HERO ID: 7768

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 14:

Test Model

High

Test models were described in detail and appropriate

for the endpoints assessed.

Metric 15:

Number per Group

Low

There was only one study group for each of the three
exposure concentrations tests (i.e., no replicates).

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

The assessment methodology addressed the intended
outcomes of interest.

Metric 17:

Consistency of Outcome Assessment

High

Outcome assessment protocol was consistent across
study groups.

Metric 18:

Sampling Adequacy

High

The number of cells/dose was reported and is appro-
priate (50 cells/dose).

Metric 19:

Blinding of Assessors

High

Test substance was supplied under code; assessors
did not know its identity until after scoring.

Domain 6: Confounding / Variable Control

Metric 20:

Confounding Variables in Test Design and
Procedures

High

There were no confounding variables in test design
or procedures that were reported by study authors.

Metric 21:

Confounding Variables in Outcomes Unre-
lated to Exposure

High

There were no confounding variables reported unre-
lated to exposure.

Domain 7: Data Presentation and Analysis

Metric 22:

Data Analysis

High

Statistical analyses were clearly described and pre-
sented in results tables.

Metric 23:

Data Interpretation

High

Data were reported in such a way as to allow inter-
pretation of test results.

Metric 24:

Cytotoxicity Data

Medium

Metric 25:

Reporting of Data

High

Data were presented for percent cells with aberra-
tions in three ways for each exposure concentration:
total, simple, and complex aberrations.

Overall Quality Determination

High

1.2

Extracted

Yes

Continued on next page . ..

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.. . continued from previous page

Study Citation:

Data Type:
HERO ID:

S. M. Galloway, M. J. Armstrong, C. Reuben, S. Colman, B. Brown, C. Cannon, A. D. Bloom, F. Nakamura, M. Ahmed, S. Duk, J.
Rimpo, B. H. Margolin, M. A. Resnick, B. Anderson, E. Zeiger (1987). Chromosome aberrations and sister chromatid exchanges in
Chinese hamster ovary cells: evaluations of 108 chemicals Environmental and Molecular Mutagenesis, 10(Suppl. 10,Suppl. 10), 1-175
1,4-Dioxane in vitro SCE
7768

Domain

Metric

Rating^ MWF* Score

Comments^

MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; X MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

This metric met the criteria for high confidence as expected for this type of study

Page 117 of 187

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Table 40: In vitro evaluation results of Haworth et al 1983 forbacterial reverse mutation study

Study Citation: S. Haworth, T. Lawlor, K. Mortelmans, W. Speck, E. Zeiger (1983). Salmonella mutagenicity test results for 250 chemicals Environ-
mental Mutagenesis, 5(Suppl l,Suppl 1), 3-142
Data Type: Bacterial reverse mutation for 1,4-dioxane

HERO ID: 28947

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

The test substance was identified as 1,4-dioxane with

the correct CASRN.

Metric 2:

Test Substance Source

High

The commercial source of the test substance was re-

ported, including manufacturer lot number.

Metric 3:

Test Substance Purity

Medium

The test substance was reported to be "Purified"

according to the manufacturer label.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

Appropriate concurrent negative control groups were

included (water).

Metric 5:

Positive Controls

High

Positive controls were tested concurrently with each

test substance. The identity of each positive control

was reported and appropriate for different strains

with and without metabolic activation. Positive con-

trols yielded positive results.

Metric 6:

Assay Procedures

High

Assay methods and procedures were described in de-

tail and were applicable to the study type.

Metric 7:

Standards for Tests

Not Rated

This metric is not applicable to this study type.

Domain 3: Exposure Characterization

Metric 8:

Preparation and Storage of Test Substance

High

Test substance preparation was reported. Test sub-

stance storage was not reported (single-dose admin-

istration).

Metric 9:

Consistency of Exposure Administration

High

Exposure administration was consistent across treat-

ment groups.

Metric 10:

Reporting of Doses/Concentrations

High

The doses were reported without ambiguity.

Metric 11:

Number of Exposure Groups and Concentra-

High

The exposure duration for the pre-incubation proto-

tion Spacing

col was reported and appropriate.

Metric 12:

Exposure Route and Method

High

The maximum dose was chosen based on solubil-

ity limits or cytotoxicity. The number of exposure

groups and dose spacing was reported and appropri-

ate for this assay (100, 333.3, 1000, 3333.3, or 10000

Pg/ plate).

Continued on

next page . .

Page 118 of 187

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.. . continued from previous page

HERO ID: 28947

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 13:

Metabolic Activation

Medium

X 1

The source and method of preparation of the rat
liver S9 fraction was reported; however, the concen-
tration of S9 in the bacterial mutagenicity assay was
not specified.

Domain 4: Test Model

Metric 14:

Test Model

High

x 2

The identity and donor source of the bacterial

strains used here were identified, and these strains
are routinely used for the outcome of interest. It
was noted that the cultures were "routinely checked
for genetic integrity as recommended by Ames et al.
(1975)."

Metric 15:

Number per Group

High

x 1

Each assay was plated in triplicate.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

x 2

The outcome assessment methodology is appropriate
for the outcome of interest.

Metric 17:

Consistency of Outcome Assessment

High

x 1

The outcome assessment was consistent across treat-
ment groups.

Metric 18:

Sampling Adequacy

Not Rated

This metric is not applicable to this endpoint.

Metric 19:

Blinding of Assessors

Not Rated

Number of colonies is an objective outcome and

blinding assessors is not necessary; however, the
identity of each test substance assessed in this study
was coded and not known to the assessors.

Domain 6: Confounding / Variable Control

Metric 20: Confounding Variables in Test Design and High x 2 2 No differences among treatment group parameters

Procedures were reported.

Metric 21: Confounding Variables in Outcomes Unre- High x 1 1 No confounding variables were reported,

lated to Exposure
Domain 7: Data Presentation and Analysis

Metric 22: Data Analysis High X 1 1 A positive result was defined as a "reproducible,

dose-related increase, whether it be twofold over
background or not." Therefore, no statistical analy-
sis was reported directly in the study; however, this
is appropriate for this study design. Raw data are
provided and could be analyzed independently.

Metric 23: Data Interpretation High X 2 2 Evaluation criteria (number of colonies) was re-

ported and consistent with current standards.

Continued on next page . ..

Page 119 of 187

-------
. continued from previous page

Study Citation: S. Haworth, T. Lawlor, K. Mortelmans, W. Speck, E. Zeiger (1983). Salmonella mutagenicity test results for 250 chemicals Environ-
mental Mutagenesis, 5(Suppl l,Suppl 1), 3-142
Data Type:	Bacterial reverse mutation for 1,4-dioxane

HERO ID:	28947

Domain Metric

Rating^

MWF*

Score

Comments^

Metric 24: Cytotoxicity Data

High

X 1

1

A dose-setting experiment was conducted to assess
cytotoxicity levels (viability, reduced numbers of
colonies). If toxicity was observed in the prelimi-
nary experiment, the doses for the mutagenicity as-
say were selected so that the highest dose exhibited
some degree of toxicity.

Metric 25: Reporting of Data

High

x 2

2

All data are adequately reported.

Overall Quality Determination1"

High



1.1



Extracted

Yes









MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; x MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 120 of 187

-------
Table 41: In vitro evaluation results of Goldsworthy et al 1991 for carcinogenicity in rat nasal epithelial cells and hepatocytes study

HERO ID: 62925

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Test substance identified as "1,4-dioxane".

Metric 2:

Test Substance Source

High

The source of the test substance was reported. The

batch/lot number was not reported, but the test sub-
stance is not expected to vary in composition.

Metric 3:

Test Substance Purity

High

Test substance reported to be of HPLC grade, 99.9%
purity.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

A concurrent media control was utilized.

Metric 5:

Positive Controls

High

Two positive control groups were included in this
study (2-Acetylaminofluorene dissolved in DMSO
and dimethylnitrosamine).

Metric 6:

Assay Procedures

Medium

Details on duration of cell incubation, medium, and

use of a radioactive nucleoside were reported. Other
details on test conditions are not reported.

Metric 7:

Standards for Tests

Not Rated

Not applicable.

Domain 3: Exposure Characterization

Metric 8:

Preparation and Storage of Test Substance

Medium

Preparation and storage of the test substance was
not reported, but information on solubility of test
substance suggests unlikely impact on results.

Metric 9:

Consistency of Exposure Administration

High

Cells were exposed in same culture medium for con-
sistent lengths of time.

Metric 10:

Reporting of Doses/Concentrations

High

The exposure concentrations were reported as point
estimates.

Metric 11:

Number of Exposure Groups and Concentra-
tion Spacing

High

Exposure duration was reported and appropriate for
this study type.

Metric 12:

Exposure Route and Method

High

The number of exposure groups and concentration
spacing were adequate to evaluate a dose-response.

Metric 13:

Metabolic Activation

High

Some groups included hepatocytes collected from
rats pretreated with test substance to provide the
opportunity for enzyme induction.

Domain 4: Test Model

Metric 14:

Test Model

High

The test model and descriptive information were re-

ported and appropriate.

Continued on next page . ..

Page 121 of 187

-------
.. . continued from previous page

HERO ID: 62925

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 15:

Number per Group

High

The number of replicates per group were reported

and appropriate.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

The outcome assessment methodology was appropri-

ate for this endpoint.

Metric 17:

Consistency of Outcome Assessment

High

Details of the outcome assessment were reported and

outcomes were assessed consistently across study

groups.

Metric 18:

Sampling Adequacy

High

Adequate sampling (25 cells scored for each of 3

Metric 19:

Blinding of Assessors

Not Rated

The outcome assessment relied on quantitative au-

toradiography. Blinding is not a concern in this

study.

Domain 6: Confounding / Variable Control

Metric 20:

Confounding Variables in Test Design and

High

No differences in test design and procedures were

Procedures

reported that would significantly influence the out-

come assessment.

Metric 21:

Confounding Variables in Outcomes Unre-

High

There were no reported differences among the study

lated to Exposure

replicates unrelated to exposure.

Domain 7: Data Presentation and Analysis

Metric 22:

Data Analysis

High

Statistical methods were clearly described and pre-

sented.

Metric 23:

Data Interpretation

High

The study authors described the evaluation criteria

for the test and noted these were consistent with the

cited standard protocol.

Metric 24:

Cytotoxicity Data

Medium

The methods of measurement were not fully de-

scribed, but signs of toxicity were noted in the data

table.

Metric 25:

Reporting of Data

High

Data were reported for all outcomes and groups.

Overall Quality Determination

High

1.1

Extracted

Yes

Continued on next page . ..

Page 122 of 187

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. continued from previous page

Study Citation: T. L. Goldsworthy, T. M. Monticello, K. T. Morgan, E. Bermudez, D. M. Wilson, R. Jackh,Butterworth BE (1991). Examination of
potential mechanisms of carcinogenicity of 1,4-dioxane in rat nasal epithelial cells and hepatocytes Archives of Toxicology, 65(1,1), 1-9
Data Type:	Goldsworthy et al. 1991 in vitro hepatocyte DNA repair

HERO ID:	62925

Domain	Metric	Rating^ MWF* Score	Comments^

* MWF = Metric Weighting Factor

t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

+ The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

if any metric is Unacceptable

Overall rating =

J]. (Metric Score; x MWF;) / J] . MWFj

(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 123 of 187

-------
Table 42: In vitro evaluation results for Woo et al 1977 for DNA binding assay study

Study Citation: Y. T. Woo, M. F. Argus, J. C. Arcos (1977). Tissue and subcellular distribution of 3H-dioxane in the rat and apparent lack of

microsome-catalyzed covalent binding in the target tissue Life Sciences, 21(10,10), 1447-1456
Data Type: DNA binding assay

HERO ID: 62950

Domain

Metric

Rating^

MWF* Score

Comments^

Domain 1: Test Substance

Metric 1: Test Substance Identity

Metric 2: Test Substance Source
Metric 3: Test Substance Purity

High X 2 2 The test substance was identified as p-dioxane (1,4-

dioxane). The dioxane was tritiated to trace radioac-
tivity and referred to as 3H-dioxane throughout the
study.

High X 1 1 The commercial source of the test substance was re-

ported.

Medium X 1 2 The test substance was reported to be "of analytical

or reagent grade." The purity was not reported, but
this is not considered to have affected the results.

Domain 2: Test Design

Metric 4: Negative and Vehicle Controls

Metric 5: Positive Controls

Metric 6: Assay Procedures

Metric 7: Standards for Tests

High
High
High

Not Rated

x 2

x 1
NA

The negative control conditions for this experiment
were the complete test system less the microsomes
or NADPH system.

Benzo[a]pyrene was included under the same con-
ditions as a positive control. Positive results were
observed under the positive control conditions.
Assay procedures were described adequately and
were appropriate for the endpoint of interest.

This metric is not applicable to this study type.

Domain 3: Exposure Characterization

Metric 8: Preparation and Storage of Test Substance Medium

Metric 9: Consistency of Exposure Administration High

Metric 10: Reporting of Doses/Concentrations High

Metric 11: Number of Exposure Groups and Concentra- High
tion Spacing

Metric 12: Exposure Route and Method High

X 1 2 The preparation of the test substance was briefly

described. The storage of the test substance was
not described.

X 1 1 Exposure administration was reported to be consis-

tent among treatment groups.

X 2 2 Doses were reported in terms of radioactivity of the

3H-dioxane (82 pCi). Doses in mg/kg-bw can be
calculated based on radioactivity of the 3H-dioxane
(8.6 Ci/mmole). Therefore, doses were reported ad-
equately.

X 2 2 Exposure duration was appropriate for the outcome

of interest.

X 1 1 There was only one exposure group, but the dose was

considered adequate for the outcome of interest.

Continued on next page

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.. . continued from previous page

Study Citation: Y. T. Woo, M. F. Argus, J. C. Arcos (1977). Tissue and subcellular distribution of 3H-dioxane in the rat and apparent lack of

microsome-catalyzed covalent binding in the target tissue Life Sciences, 21(10,10), 1447-1456
Data Type: DNA binding assay

HERO ID: 62950

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 13:

Metabolic Activation

High

X 1

In cases where, primary liver cells were harvested

from rats following pretreatment with inducers of

microsomal mixed function oxidases (phenobarbital,

3-methylchloanthrene, PCBs)

Domain 4: Test Model

Metric 14:

Test Model

High

x 2

The test model, calf thymus DNA, was reported but

no other details were provided.

Metric 15:

Number per Group

Unacceptable

x 1

The replicates per study group were not reported.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

x 2

The outcome assessment methodology was appropri-

Metric 17:

Consistency of Outcome Assessment

Medium

x 1

The positive control, benzo[a]pyrene, was not tested

under all conditions that the dioxane was tested un-

der (excluded +cytosol condition). Otherwise, the

outcome assessment was reported to be consistent.

Metric 18:

Sampling Adequacy

Not Rated

This metric is not applicable to the study design.

Metric 19:

Blinding of Assessors

Not Rated

This metric is not applicable to the study type.

Domain 6: Confounding / Variable Control

Metric 20:

Confounding Variables in Test Design and

High

x 2

No confounding variables in the study design were

Procedures

reported.

Metric 21:

Confounding Variables in Outcomes Unre-

High

x 1

No confounding variables in outcomes unrelated to

lated to Exposure

exposure were reported.

Domain 7: Data Presentation and Analysis

Metric 22:

Data Analysis

Unacceptable

x 1

No statistics were provided, because it appears that

n = 1 for all test conditions..

Metric 23:

Data Interpretation

High

x 2

The data were interpreted appropriately.

Metric 24:

Cytotoxicity Data

Not Rated

This metric is not applicable to the study design, as

no cells were utilized.

Metric 25:

Reporting of Data

Medium

x 2

Results were reported for 3H-dioxane treatment only

(no results reported for 14C-dioxane treatment).

Overall Quality Determination'

Unacceptable*^

1.3

Extracted

Continued on next page . ..

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. continued from previous page

Study Citation: Y. T. Woo, M. F. Argus, J. C. Arcos (1977). Tissue and subcellular distribution of 3H-dioxane in the rat and apparent lack of

microsome-catalyzed covalent binding in the target tissue Life Sciences, 21(10,10), 1447-1456
Data Type:	DNA binding assay

HERO ID:	62950

Domain	Metric	Rating^ MWF* Score	Comments^

** Consistent with our Application of Systematic Review in TSCARisk Evaluations document, if a metric for a data source receives a score of Unacceptable (score = 4),
EPA will determine the study to be unacceptable. In this case, one or more of the metrics were rated as unacceptable. As such, the study is considered unacceptable and
the score is presented solely to increase transparency.

* MWF = Metric Weighting Factor

t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

+ The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

if any metric is Unacceptable

Overall rating =

J]. (Metric Score; x MWF;) / J] . MWFj

(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 126 of 187

-------
Table 43: In vitro evaluation results of Nestmann et al 1984 for Ames test study

Study Citation: E. R. Nestmann, R. Otson, D. J. Kowbel, P. D. Bothwell, T. R. Harrington (1984). Mutagenicity in a modified Salmonella assay of

fabric-protecting products containing 1,1,1-trichloroethane Environmental and Molecular Mutagenesis, 6(1,1), 71-80
Data Type: 1,4-D Ames test

HERO ID: 194339

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

x 2

The test substance was identified by name ("p-

dioxane") in the study, though it was not described
in any detail other than as a component of the 2
fabric protectors being evaluated.

Metric 2:

Test Substance Source

Medium

x 1

The specific source of 1,4-dioxane was not stated in
the paper, though the authors noted that "standards
were obtained from Fisher Scientific Co., Limited,
and Aldrich Chemical Co." Lot numbers were pro-
vided for TCE from these 2 sources. It is assumed
that a standard of 1,4-dioxane was obtained from
these sources. However, the uncertainty regarding
the source of the test substance is not likely to im-
pact the results of the study.

Metric 3:

Test Substance Purity

Low

x 1

Purity and grade of test substance were not re-

ported. However, GC and GC-MS analyses were de-
scribed in detail.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

x 2

A "no-dose" control (also referred to in the study
as a "control (blank) chamber") was included in the
study.

Metric 5:

Positive Controls

Medium

x 2

Four positive controls were employed and results
shown on data summary tables, though they were
not discussed in the text.

Metric 6:

Assay Procedures

Medium

x 1

Study authors cite methods described in Ames et

al. (1975) and obtained the tester strains from the
Ames lab. Study authors noted a test deviation (not
incorporating test substances into the top agar but
rather adding them to open Petri dishes in dessica-
tors containing the culture dishes).

Metric 7:

Standards for Tests

Not Rated

Not applicable to this study design.

Domain 3: Exposure Characterization

Metric 8:

Preparation and Storage of Test Substance

Low

x 1

Study describes preparation and storage of gaseous
standards of test substances and general prepara-
tion of liquid samples added to culture dishes (it is
assumed that these include 1,4-dioxane).

Metric 9:

Consistency of Exposure Administration

High

x 1

Exposure administration was consistent across study
groups.

Continued on next page . ..

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-------
. continued from previous page

Study Citation: E. R. Nestmann, R. Otson, D. J. Kowbel, P. D. Bothwell, T. R. Harrington (1984). Mutagenicity in a modified Salmonella assay of

fabric-protecting products containing 1,1,1-trichloroethane Environmental and Molecular Mutagenesis, 6(1,1), 71-80
Data Type: 1,4-D Ames test

HERO ID: 194339

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 10:

Reporting of Doses/Concentrations

High

x 2

Nominal concentrations and time-weighted average
exposure levels were reported for each exposure
group.; air concentrations were measured and re-
ported analytically

Metric 11:

Number of Exposure Groups and Concentra-
tion Spacing

Low

x 2

Incubation period was 24 hours exposure to test sub-
stance, followed by an additional 24 hours prior to

scoring plates. The plate incorporation method re-
quires a 48-72 hour exposure.

Metric 12:

Exposure Route and Method

Low

x 1

Only 2 of 5 Salmonella strains were exposed to test
substance, and only 3 exposure concentrations were
employed for 1,4-dioxane.

Metric 13:

Metabolic Activation

Medium

x 1

Use of common metabolic activation system was re-
ported, though not described in much detail.

Domain 4: Test Model

Metric 14:

Test Model

Medium

x 2

Study employed commonly used bacterial strains
and reported their source, but cited Ames et al.
(1975) for a detailed description of them.

Metric 15:

Number per Group

Low

x 1

Study employed 2 replicates/strain of bacteria. Ini-
tial bacterial cell counts were not reported.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

x 2

Outcome assessment methodology reported the in-
tended outcomes of interest.

Metric 17:

Consistency of Outcome Assessment

High

x 1

Outcome assessment was carried out consistently
across study groups.

Metric 18:

Sampling Adequacy

Not Rated

Not applicable to mutagenicity assays

Metric 19:

Blinding of Assessors

Not Rated

Not applicable (no subjective outcomes were as-
sessed)

Domain 6: Confounding / Variable Control

Metric 20:

Confounding Variables in Test Design and
Procedures

High

x 2

No potential confounding variables were reported.

Metric 21:

Confounding Variables in Outcomes Unre-
lated to Exposure

High

x 1

No confounding variables unrelated to exposure were
reported.

Domain 7: Data Presentation and Analysis

Metric 22: Data Analysis Low X 1 3 data interpretation was limited to calculating means

of duplicate plates; means were considered different
from background if there was a two-fold differences
but not statistical analysis was performed.

Continued on next page . ..

Page 128 of 187

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. continued from previous page

Study Citation: E. R. Nestmann, R. Otson, D. J. Kowbel, P. D. Bothwell, T. R. Harrington (1984). Mutagenicity in a modified Salmonella assay of

fabric-protecting products containing 1,1,1-trichloroethane Environmental and Molecular Mutagenesis, 6(1,1), 71-80
Data Type:	1,4-D Ames test

HERO ID:	194339

Domain



Metric

Rating^

MWF*

Score

Comments^



Metric 23:

Metric 24:
Metric 25:

Data Interpretation

Cytotoxicity Data
Reporting of Data

Low

Not Rated
Low

x 2

NA
x 2

6

NA
6

plates were scored for mutant colonies and back-
ground rates, but details of scoring methods were
not provided

Study did not evaluate cytotoxicity.

Data were reported as revertants/plate for each ex-
posure group, but data are insufficient to perform
any statistical analysis (the only data reported is
the mean of the duplicate plates).

Overall Quality Determination*



Medium



1.9



Extracted





Yes









MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; x MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating,
ft This metric met the criteria for high confidence as expected for this type of study

Page 129 of 187

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Table 44: In vitro evaluation results of Zimmermann et al 1985 (194343) for an aneuploidy study in Saccharomyces cerevisiae

Study Citation: Zimmermann, FK; Mayer, VW; Scheel, I; Resnick, MA (1985). Acetone, methyl ethyl ketone, ethyl acetate, acetonitrile and other
polar aprotic solvents are strong inducers of aneuploidy in Saccharomyces cerevisiae Mutation Research, 149(3), 339-351

Data Type:

HERO ID: 194343

Domain Metric Rating^ MWF* Score Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

Unacceptable

Test substance is not clearly identified (it was re-
ferred to as dioxane in the study).

Metric 2:

Test Substance Source

Unacceptable

Test substance source was not reported.

Metric 3:

Test Substance Purity

Unacceptable

Test substance purity was not reported.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

Low

A negative control was used, but not described

(identity for the experiment referred to as dioxane
was not reported).

Metric 5:

Positive Controls

High

A positive control was used.

Metric 6:

Assay Procedures

Unacceptable

Assay methods were not reported for the study re-
ferred to as dioxane.

Metric 7:

Standards for Tests

Unacceptable

QC criteria were not specifically reported for the
dioxane study.

Domain 3: Exposure Characterization

Metric 8:

Preparation and Storage of Test Substance

Unacceptable

No information was provided on the preparation and
storage.

Metric 9:

Consistency of Exposure Administration

Unacceptable

Critical exposure details were not reported for the
dioxane study.

Metric 10:

Reporting of Doses/Concentrations

Unacceptable

Exposure concentrations were reported for the study
for dioxane.

Metric 11:

Number of Exposure Groups and Concentra-
tion Spacing

High

Exposure duration was acceptable for this type of
study.

Metric 12:

Exposure Route and Method

High

The number of exposure groups was reported. Spac-
ing was acceptable.

Metric 13:

Metabolic Activation

Unacceptable

No information was provided on metabolic activa-
tion.

Domain 4: Test Model

Metric 14:

Test Model

High

The test model (yeast) was reported and was accept-
able for evaluating mutagenicity.

Metric 15:

Number per Group

Unacceptable

The number of replicates per group was not reported
for the dioxane study.

Domain 5: Outcome Assessment

Continued on next page . ..

Page 130 of 187

-------
... continued from previous page

Data Type:

HERO ID: 194343

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 16:

Outcome Assessment Methodology

High

The outcome assessment methodology was reported

and was appropriate for the outcome of interest.

Metric 17:

Consistency of Outcome Assessment

Low

There were no evidence inconsistencies among the

exposure groups; however, there were few descriptive

details for the dioxane study.

Metric 18:

Sampling Adequacy

Low

Limitations were reported for sampling although the

duration following treatment was reported.

Metric 19:

Blinding of Assessors

Unacceptable

Blinding was not reported.

Domain 6: Confounding / Variable Control

Metric 20:

Confounding Variables in Test Design and

Low

There were no confounding variables reported but

Procedures

based on limited details confidence is low.

Metric 21:

Confounding Variables in Outcomes Unre-

Low

No outcomes unrelated to exposure were reported,

lated to Exposure

but based on limited details confidence is low.

Domain 7: Data Presentation and Analysis

Metric 22:

Data Analysis

Low

Data were provided. Few/no details regarding con-

duct of statistical analysis was provided.

Metric 23:

Data Interpretation

Low

There was indication that scoring and/or evaluation

criteria were not consistent with current guidelines;

however, few details were provided so confidence is

Metric 24:

Cytotoxicity Data

Unacceptable

Cytotoxicity was not defined or presented.

Metric 25:

Reporting of Data

Low

Data that were presented were acceptable to demon-

strate a negative result; however, few details were

provided so confidence is low.

Overall Quality Determination1

Unacceptable**

3.5

Extracted

Yes

MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

if any metric is Unacceptable

Overall rating =

J]. (Metric Score; x MWF;) / ]T\ MWFj

(round to the nearest tenth) otherwise

^ This metric met the criteria for high confidence as expected for this type of study

Page 131 of 187

-------
Table 45: Animal toxicity evaluation results of Goldsworthy et al 1991 for gavage study in rats on hepatocyte cell proliferation

HERO ID: 62925

Domain

Metric

Rating^ MWF* Score

Comments^

Domain 1: Test Substance
Metric 1:
Metric 2:

Test Substance Identity
Test Substance Source

Metric 3: Test Substance Purity

High X 2 2 Test substance identified as "1,4-dioxane".

High X 1 1 The source of the test substance was reported. The

batch/lot number was not reported, but the test sub-
stance is not expected to vary in composition.

High X 1 1 Test substance was reported to be of HPLC grade,

99.9% purity.

Domain 2: Test Design

Metric 4: Negative and Vehicle Controls

Metric 5: Positive Controls

Metric 6: Randomized Allocation

High X 2 2 Concurrent negative controls were utilized (water).

Not Rated NA NA No positive control group was needed for this study

type.

Low X 1 3 The study did not report how animals were allocated

to study groups.

Domain 3: Exposure Characterization

Metric 7: Preparation and Storage of Test Substance

Medium

x 1

The study notes that the test substance was admin-
istered in water and the test substance is known to
be soluble in water. Storage conditions were not re-
ported.

Metric 8:

Consistency of Exposure Administration

High

Consistent gavage volumes were administered.

Metric 9:

Reporting of Doses/Concentrations

High

Gavage doses were reported.

Metric 10:

Exposure Frequency and Duration

High

The exposure frequency and duration (single dose)
was reported and appropriate for this endpoint.

Metric 11:

Number of Exposure Groups and Dose Spac-
ing

High

A single dose level was utilized, but this was consid-
ered adequate for evaluating hepatocyte cell replica-
tion at different time points compared to controls.

Metric 12:

Exposure Route and Method

High

The route of exposure was appropriate for this end-
point.

Domain 4: Test Organism

Metric 13:

Test Animal Characteristics

High

Test animal characteristics were reported.

Metric 14:

Adequacy and Consistency of Animal Hus-
bandry Conditions

High

Animal husbandry conditions were adequate and
consistent across control and exposed groups.

Continued on

next page .

Page 132 of 187

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. continued from previous page

Study Citation: T. L. Goldsworthy, T. M. Monticello, K. T. Morgan, E. Bermudez, D. M. Wilson, R. Jackh,Butterworth BE (1991). Examination of
potential mechanisms of carcinogenicity of 1,4-dioxane in rat nasal epithelial cells and hepatocytes Archives of Toxicology, 65(1,1), 1-9
Data Type:	Gavage Study - Hepatocyte Cell Proliferation

HERO ID:	62925

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 15:

Number per Group

Medium

X

1

2

The number of animals in the exposed treatment
groups was adequate for the outcome analysis (n =
5), but a smaller number of animals was included in
the negative control group (n = 3) without reference
to a historical dataset.

Domain 5: Outcome Assessment











Metric 16:

Outcome Assessment Methodology

High

X

2

2

The outcome assessment methodology was appropri-
ate for this endpoint.

Metric 17:

Consistency of Outcome Assessment

High

X

1

1

The outcome assessment as consistent for all groups.

Metric 18:

Sampling Adequacy

High

X

1

1

The number of hepatocyte nuclei (n=2,000) from
each liver section was adequate for the outcome of
interest.

Metric 19:

Blinding of Assessors

Not Rated

NA

NA

The outcome assessment relied on quantitative au-













toradiography. Blinding is not a concern in this
study.

Metric 20:

Negative Control Response

High

X

1

1

The control response was adequate.

Domain 6: Confounding / Variable Control











Metric 21:

Confounding Variables in Test Design and
Procedures

Medium

X

2

4

The study did not report on initial body weights
or food/water intake during this particular study,
but this is not likely to have a significant impact on
results.

Metric 22:

Health Outcomes Unrelated to Exposure

High

X

1

1

No health outcomes or deaths were reported in the











study.

Domain 7: Data Presentation and Analysis











Metric 23:

Statistical Methods

High

X

1

1

Statistical methods were reported and appropriate
for the dataset.

Metric 24:

Reporting of Data

High

X

2

2

Data were reported for all outcomes and groups.

Overall Quality Determination"



High





1.2



Extracted



Yes











MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; x MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 133 of 187

-------
Table 46: In vitro evaluation results of Mcgregor et al 1991 for mice lymph cell mutation assay study

Study Citation: D. B. Mcgregor, A. G. Brown, S. Howgate, D. Mcbride, C. Riach, W. J. Caspary (1991). Responses of the L5178Y mouse lymphoma

cell forward mutation assay. V: 27 coded chemicals Environmental and Molecular Mutagenesis, 17(3,3), 196-219
Data Type: 1,4-D Mouse Lymph Cell Mutation Assay

HERO ID: 194381

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Test substance was identified by established name,

CASRN, and chemical structure.

Metric 2:

Test Substance Source

High

The source of the test substance was identified in
the report.

Metric 3:

Test Substance Purity

Low

Test substance purity was not reported.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

Fischer's medium without serum was used as a ve-

hicle control.

Metric 5:

Positive Controls

High

Two positive controls were used; their response was
appropriate (significant increase in mutation fre-
quency).

Metric 6:

Assay Procedures

High

Assay procedures were well described.

Metric 7:

Standards for Tests

High

The paper followed quality control guidelines and
response criteria described in Caspary 1988.

Domain 3: Exposure Characterization

Metric 8:

Preparation and Storage of Test Substance

Medium

General information regarding test substance prepa-
ration was included, but storage conditions were not
provided.

Metric 9:

Consistency of Exposure Administration

High

Information on exposure administration was re-
ported and consistency of administration is inferred
from the text.

Metric 10:

Reporting of Doses/Concentrations

High

Exposure concentrations were reported for each of
the trials.

Metric 11:

Number of Exposure Groups and Concentra-
tion Spacing

High

Exposure duration for each phase of the testing was
clearly stated and appropriate for the endpoint.

Metric 12:

Exposure Route and Method

High

Exposure groups and concentration spacing was
based on initial toxicity testing and is considered
adequate.

Metric 13:

Metabolic Activation

High

Trials were run with and without metabolic activa-

tion. Preparation of S9 was described in detail.

Domain 4: Test Model

Metric 14:

Test Model

High

Test model was described and is appropriate.

Continued on

next page .

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.. . continued from previous page

Study Citation: D. B. Mcgregor, A. G. Brown, S. Howgate, D. Mcbride, C. Riach, W. J. Caspary (1991). Responses of the L5178Y mouse lymphoma

cell forward mutation assay. V: 27 coded chemicals Environmental and Molecular Mutagenesis, 17(3,3), 196-219
Data Type: 1,4-D Mouse Lymph Cell Mutation Assay

HERO ID: 194381

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 15:

Number per Group

High

X 1

The number of cells/culture was reported, as well as
the number of replicate cultures/exposure concen-
tration. They are appropriate for the study type.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

x 2

The assessment methodology addressed the intended
outcomes of interest.

Metric 17:

Consistency of Outcome Assessment

Medium

x 1

Outcome assessment protocol was consistent across

study groups.

Metric 18:

Sampling Adequacy

Not Rated

Not applicable

Metric 19:

Blinding of Assessors

Not Rated

Automated colony counting was employed and chem-
icals were coded during the study.

Domain 6: Confounding / Variable Control

Metric 20:

Confounding Variables in Test Design and
Procedures

High

x 2

There were no confounding variables in test design
or procedures that were reported by study authors.

Metric 21:

Confounding Variables in Outcomes Unre-
lated to Exposure

Medium

x 1

Authors reported that one of the positive control cul-
tures was contaminated, but data from the remain-
ing exposure replicates or groups were valid. The
trial containing this culture was not reported in fi-
nal results tables as it failed to meet quality control
criteria of the study.

Domain 7: Data Presentation and Analysis

Metric 22:

Data Analysis

High

x 1

Statistical evaluations were clearly described and
presented in results tables.

Metric 23:

Data Interpretation

High

x 2

Data were reported in such a way as to allow inter-
pretation of test results.

Metric 24:

Cytotoxicity Data

High

x 1

Cytotoxicity test was described by the study authors
as the first step in evaluation, to determine the ex-
posure concentrations for the test substance.

Metric 25:

Reporting of Data

High

x 2

Data were presented for all outcomes by exposure
group.

Overall Quality Determination

High

1.2

Extracted

Yes

Continued on next page . ..

Page 135 of 187

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. continued from previous page

Study Citation: D. B. Mcgregor, A. G. Brown, S. Howgate, D. Mcbride, C. Riach, W. J. Caspary (1991). Responses of the L5178Y mouse lymphoma

cell forward mutation assay. V: 27 coded chemicals Environmental and Molecular Mutagenesis, 17(3,3), 196-219
Data Type:	1,4-D Mouse Lymph Cell Mutation Assay

HERO ID:	194381

Domain	Metric	Rating^ MWF* Score	Comments^

* MWF = Metric Weighting Factor

t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

+ The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

if any metric is Unacceptable

Overall rating =

J]. (Metric Score; x MWF;) / J] . MWFj

(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 136 of 187

-------
Table 47: In vitro evaluation results of Hellmer and Bolcsfoldi 1992 for DNA repair in E. coli study

Study Citation: L. Hellmer, G. Bolcsfoldi (1992). An evaluation of the E. coli K-12 uvrB/recA DNA repair host-mediated assay: I. In vitro sensitivity

of the bacteria to 61 compounds Mutation Research, 272(2,2), 145-160
Data Type: 1,4-D in vitro DNA repair test in E. coli

HERO ID: 194717

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

x 2

The test substance was identified as 1,4-dioxane

Metric 2:

Test Substance Source

Medium

x 1

The source of the test substance was not specifi-
cally reported, but it was noted that the chemicals
tested were purchased from a commercial source.
The product number and batch/lot number were
also not reported; however, the material is not ex-
pected to vary in composition.

The omitted details are unlikely to have a substan-
tial impact on the results.

Metric 3:

Test Substance Purity

Low

x 1

The purity and/or grade of the test substance were

not reported. It was noted that all chemicals tested
were of the highest purity obtainable.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

Medium

x 2

Study authors report using a concurrent negative
solvent control; however, the solvent used for 1,4-
dioaxane was not specified. This limitation is un-
likely to have a substantial impact on results.

Metric 5:

Positive Controls

High

x 2

A positive control (4-nitroquinoline-N-oxide) was
used for tests without S9 metabolic activation; no
positive control was used for tests with the S9
metabolic activation.

Metric 6:

Assay Procedures

Medium

x 1

Methods and procedures were partially described,
but appear to be appropriate.

Metric 7:

Standards for Tests

Not Rated

Not applicable for this study

Domain 3: Exposure Characterization

Metric 8:

Preparation and Storage of Test Substance

High

The test substance preparation was reported; the
solutions were made immediately before the experi-
ment and did not need to be stored.

Metric 9:

Consistency of Exposure Administration

High

Exposures were reported to be administered consis-
tently across study groups.

Metric 10:

Reporting of Doses/Concentrations

High

The highest concentration was reported; there were
no effects at this concentration.

Metric 11:

Number of Exposure Groups and Concentra-

High

The exposure duration was reported (1 day)

tion Spacing

Continued on next page . ..

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.. . continued from previous page

Study Citation: L. Hellmer, G. Bolcsfoldi (1992). An evaluation of the E. coli K-12 uvrB/recA DNA repair host-mediated assay: I. In vitro sensitivity

of the bacteria to 61 compounds Mutation Research, 272(2,2), 145-160
Data Type: 1,4-D in vitro DNA repair test in E. coli

HERO ID: 194717

Domain Metric Rating^ MWF* Score Comments^

Metric 12:

Exposure Route and Method

Medium

x 1

The number of exposure groups and
dose/concentration spacing were justified by
study authors (diluted in 7 half log steps or 2-fold
dilution steps; but only the highest concentration
was reported. The number of exposure concentra-
tions is unclear; because there were no effects at
the highest concentration, it is unlikely to have a
substantial impact on results.

Metric 13:

Metabolic Activation

High

x 1

Exposures were conducted in the presence and ab-

sence of a metabolic activation system. The source
and method of preparation were reported.

Domain 4: Test Model

Metric 14:

Test Model

High

x 2

The test models and source were reported and ap-
propriate for the outcome of interest.

Metric 15:

Number per Group

Low

x 1

The volume of bacterial mix was reported. One plate
per concentration was tested.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

x 2

The outcome assessment methodologies were appro-
priate for the endpoints of interest.

Metric 17:

Consistency of Outcome Assessment

High

x 1

The outcome assessment was carried out consis-
tently for all three experiments.

Metric 18:

Sampling Adequacy

Not Rated

Not applicable

Metric 19:

Blinding of Assessors

Not Rated

This method is not applicable to the outcome.

Domain 6: Confounding / Variable Control

Metric 20:

Confounding Variables in Test Design and
Procedures

High

x 2

There were no confounding variables noted in the
study.

Metric 21:

Confounding Variables in Outcomes Unre-
lated to Exposure

High

x 1

No confounding variable unrelated to exposure were
identified.

Domain 7: Data Presentation and Analysis

Metric 22:

Data Analysis

Medium

x 1

Statistical methods were described and appropriate

for the dataset. It was noted that the confidence
interval was determined according to the variance
for each strain from a previous experiment; this data
was not presented.

Metric 23:

Data Interpretation

High

x 2

The scoring/evaluation criteria was reported (if the
number of colonies was < 2 standard deviations of
the mean for the strains, the test was considered
significant)

Continued on next page . ..

Page 138 of 187

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... continued from previous page

Study Citation: L. Hellmer, G. Bolcsfoldi (1992). An evaluation of the E. coli K-12 uvrB/recA DNA repair host-mediated assay: I. In vitro sensitivity

of the bacteria to 61 compounds Mutation Research, 272(2,2), 145-160
Data Type:	1,4-D in vitro DNA repair test in E. coli

HERO ID:	194717

Domain Metric

Rating^

MWF*

Score

Comments^

Metric 24: Cytotoxicity Data
Metric 25: Reporting of Data

Not Rated
Medium

NA
x 2

NA
4

Cytotoxicity endpoints were not defined; however,
there was no effect at the highest concentration
tested. Cytotoxicity was not a factor in this study.

The data for the outcome was reported. The study
was negative at the highest dose tested

Overall Quality Determination1"

High



1.4



Extracted

Yes









MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; x MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 139 of 187

-------
Table 48: In vitro evaluation results of Khudoley et al 1987 for bacterial reverse mutation study

Study Citation: V. V. Khudoley, I. Mizgireuv, G. B. Pliss (1987). The study of mutagenic activity of carcinogens and other chemical agents with

Salmonella typhimurium assays: Testing of 126 compounds Archiv fur Geschwulstforschung, 57(6,6), 453-462
Data Type: Bacterial reverse mutation for 1,4-dioxane

HERO ID: 194949

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

x 2

The test substance was identified as 1,4-dioxane with

the correct CASRN.

Metric 2:

Test Substance Source

Low

x 1

The commercial source of 1,4-dioxane was not re-
ported. A subset of the 126 test substances were
reported to have been synthesized at the home in-
stitution of the authors, so it can be assumed that
the 1,4-dioxane was obtained from an unidentified
commercial source.

Metric 3:

Test Substance Purity

Low

x 1

It was reported that the "majority" of the 126 test
substances were "chemically pure". The purity of
1,4-dioxane was not reported.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

x 2

Solvent controls were included concurrently in study
design.

Metric 5:

Positive Controls

Low

x 2

Appropriate concurrent positive control test sub-
stances were included for each test condition with
and without S9 activation. Positive control data
were not reported.

Metric 6:

Assay Procedures

Low

x 1

Details of assay methods and procedures were cited

to other publications.

Metric 7:

Standards for Tests

Not Rated

This metric is not applicable to this study type.

Domain 3: Exposure Characterization

Metric

Preparation and Storage of Test Substance

Low

x 1

Assay methods were cited to other publications,
preparation and storage were not specified

Metric

Consistency of Exposure Administration

Low

x 1

Assay methods were cited to other publications.

Metric

10:

Reporting of Doses/Concentrations

Not Rated

Assay methods were cited to other publications.

Metric

11:

Number of Exposure Groups and Concentra-
tion Spacing

Low

x 2

The assay procedures were described as "routine
protocol" and cited in other references.

Metric

12:

Exposure Route and Method

Low

x 1

The number of exposure groups and dose spacing

were not reported. The assay procedures were de-
scribed as "routine protocol" and cited in other ref-
erences.

Continued on next page . ..

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-------
.. . continued from previous page

Study Citation: V. V. Khudoley, I. Mizgireuv, G. B. Pliss (1987). The study of mutagenic activity of carcinogens and other chemical agents with

Salmonella typhimurium assays: Testing of 126 compounds Archiv fur Geschwulstforschung, 57(6,6), 453-462
Data Type: Bacterial reverse mutation for 1,4-dioxane

HERO ID: 194949

Domain

Metric

Rating^ MWF* Score

Comments^

Metric 13:

Metabolic Activation

Medium

x 1

The source and method of preparation of the rat
liver S9 fraction was reported; however, the concen-
tration of S9 in the bacterial mutagenicity assay was
not specified.

Domain 4: Test Model

Metric 14:

Test Model

High

x 2

The identity and donor source of the bacterial

strains used here were identified, and these strains
are routinely used for the outcome of interest.

Metric 15:

Number per Group

Low

x 1

The number of plates per treatment group was not
reported. The assay procedures were described as
"routine protocol" and cited in other references.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

x 2

The outcome assessment methodology is appropriate
for the outcome of interest.

Metric 17:

Consistency of Outcome Assessment

High

x 1

The outcome assessment was consistent across treat-
ment groups.

Metric 18:

Sampling Adequacy

Not Rated

This metric is not applicable to this endpoint.

Metric 19:

Blinding of Assessors

Not Rated

Number of colonies is an objective outcome and
blinding assessors is not necessary.

Domain 6: Confounding / Variable Control

Metric 20:

Confounding Variables in Test Design and
Procedures

High

x 2

No differences among treatment group parameters
were reported.

Metric 21:

Confounding Variables in Outcomes Unre-
lated to Exposure

High

x 1

No confounding variables were reported.

Domain 7: Data Presentation and Analysis

Metric 22:

Data Analysis

Medium

x 1

The data were statistically analyzed, but the statis-
tical test was not reported. A positive result was de-
fined as a dose-dependent response at least 2x back-
ground mutation rates, which is appropriate for this
study design.

Metric 23:

Data Interpretation

High

x 2

Evaluation criteria (number of colonies) was re-
ported and consistent with standards and guidelines.

Metric 24:

Cytotoxicity Data

Medium

x 1

No cytotoxicity assay was included for the bacterial
mutagenicity assay; however, this is unlikely to have
a substantial impact on the study results.

Metric 25:

Reporting of Data

Low

x 2

Effect is reported as positive or negative for each
chemical, but specific data (ie specific rates of mu-
tagenicity relative to background) are not provided

Continued on next page . ..

Page 141 of 187

-------
.. . continued from previous page

Study Citation: V. V. Khudoley, I. Mizgireuv, G. B. Pliss (1987). The study of mutagenic activity of carcinogens and other chemical agents with

Salmonella typhimurium assays: Testing of 126 compounds Archiv fur Geschwulstforschung, 57(6,6), 453-462
Data Type:	Bacterial reverse mutation for 1,4-dioxane

HERO ID:	194949

Domain

Metric

Rating^ MWF* Score

Comments^

Overall Quality Determination1"

Medium

2.0

Extracted

Yes

MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; X MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

This metric met the criteria for high confidence as expected for this type of study

Page 142 of 187

-------
Table 49: In vitro evaluation results of Khudoley et al 1987 for bacterial reverse mutation study

Study Citation: V. V. Khudoley, I. Mizgireuv, G. B. Pliss (1987). The study of mutagenic activity of carcinogens and other chemical agents with

Salmonella typhimurium assays: Testing of 126 compounds Archiv fur Geschwulstforschung, 57(6,6), 453-462
Data Type: Bacterial reverse mutation for CC14

HERO ID: 194949

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

x 2

The test substance was identified as carbon tetra-

chloride with the correct CASRN.

Metric 2:

Test Substance Source

Low

x 1

The commercial source of CC14 was not reported.

A subset of the 126 test substances were reported

to have been synthesized at the home institution of

the authors, so it can be assumed that the CC14 was

obtained from an unidentified commercial source.

Metric 3:

Test Substance Purity

Low

x 1

It was reported that the "majority" of the 126 test

substances were "chemically pure". The purity of

CC14 was not reported.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

x 2

Solvent controls were included concurrently in study

design.

Metric 5:

Positive Controls

Low

x 2

Appropriate concurrent positive control test sub-

stances were included for each test condition with

and without S9 activation. Positive control data

were not reported.

Metric 6:

Assay Procedures

Not Rated

Assay methods and procedures were cited to other

publications.

Metric 7:

Standards for Tests

Not Rated

This metric is not applicable to this study type.

Domain 3: Exposure Characterization

Metric 8:

Preparation and Storage of Test Substance

Not Rated

Assay methods were cited to other publications.

Metric 9:

Consistency of Exposure Administration

Not Rated

Assay methods were cited to other publications.

Metric 10:

Reporting of Doses/Concentrations

Not Rated

Assay methods were cited to other publications.

Metric 11:

Number of Exposure Groups and Concentra-

Not Rated

The assay procedures were described as "routine

tion Spacing

protocol" and cited in other references.

Metric 12:

Exposure Route and Method

Not Rated

The number of exposure groups and dose spacing

were not reported. The assay procedures were de-

scribed as "routine protocol" and cited in other ref-
erences

Metric 13:

Metabolic Activation

Medium

x 1

The source and method of preparation of the rat

liver S9 fraction was reported; however, the concen-

tration of S9 in the bacterial mutagenicity assay was

not specified.

Continued on next page . ..

Page 143 of 187

-------
.. . continued from previous page

Study Citation: V. V. Khudoley, I. Mizgireuv, G. B. Pliss (1987). The study of mutagenic activity of carcinogens and other chemical agents with

Salmonella typhimurium assays: Testing of 126 compounds Archiv fur Geschwulstforschung, 57(6,6), 453-462
Data Type: Bacterial reverse mutation for CC14

HERO ID: 194949

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 4: Test Model

Metric 14:

Test Model

High

x 2

The identity and donor source of the bacterial

strains used here were identified, and these strains
are routinely used for the outcome of interest.

Metric 15:

Number per Group

Not Rated

The number of plates per treatment group was not
reported. The assay procedures were described as
"routine protocol" and cited in other references.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

x 2

The outcome assessment methodology is appropriate
for the outcome of interest.

Metric 17:

Consistency of Outcome Assessment

High

x 1

The outcome assessment was consistent across treat-
ment groups.

Metric 18:

Sampling Adequacy

Not Rated

This metric is not applicable to this endpoint.

Metric 19:

Blinding of Assessors

Not Rated

Number of colonies is an objective outcome and
blinding assessors is not necessary.

Domain 6: Confounding / Variable Control

Metric 20:

Confounding Variables in Test Design and
Procedures

Low

x 2

Initial conditions were not reported for each study
replicate or group.

Metric 21:

Confounding Variables in Outcomes Unre-
lated to Exposure

Low

x 1

Data on outcome differences unrelated to exposure
were not reported for each study replicate or group.

Domain 7: Data Presentation and Analysis

Metric 22:

Data Analysis

Medium

x 1

Metric 23:

Data Interpretation

High

x 2

Evaluation criteria (number of colonies) was re-
ported and consistent with standards and guidelines.

Metric 24:

Cytotoxicity Data

Not Rated

No cytotoxicity assay was included for the bacterial
mutagenicity assay; however, this is unlikely to have
a substantial impact on the study results.

Metric 25:

Reporting of Data

High

x 2

All data are adequately reported.

Overall Quality Determination

Medium

1.7

Extracted

Yes

Continued on next page

Page 144 of 187

-------
. continued from previous page

Study Citation: V. V. Khudoley, I. Mizgireuv, G. B. Pliss (1987). The study of mutagenic activity of carcinogens and other chemical agents with

Salmonella typhimurium assays: Testing of 126 compounds Archiv fur Geschwulstforschung, 57(6,6), 453-462
Data Type:	Bacterial reverse mutation for CC14

HERO ID:	194949

Domain	Metric	Rating^ MWF* Score	Comments^

* MWF = Metric Weighting Factor

t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

+ The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

if any metric is Unacceptable

Overall rating =

J]. (Metric Score; x MWF;) / J] . MWFj

(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 145 of 187

-------
Table 50:

Study Citation: V. V. Khudoley, I. Mizgireuv, G. B. Pliss (1987). The study of mutagenic activity of carcinogens and other chemical agents with

Salmonella typhimurium assays: Testing of 126 compounds Archiv fur Geschwulstforschung, 57(6,6), 453-462
Data Type: Bacterial reverse mutation for DCM

HERO ID: 194949

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

x 2

The test substance was identified as

dichloromethane with the correct CASRN.

Metric 2:

Test Substance Source

Low

x 1

The commercial source of DCM was not reported.

A subset of the 126 test substances were reported

to have been synthesized at the home institution of

the authors, so it can be assumed that the DCM was

obtained from an unidentified commercial source.

Metric 3:

Test Substance Purity

Low

x 1

It was reported that the "majority" of the 126 test

substances were "chemically pure". The purity of

DCM was not reported.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

x 2

Solvent controls were included concurrently in study

design.

Metric 5:

Positive Controls

Low

x 2

Appropriate concurrent positive control test sub-

stances were included for each test condition with

and without S9 activation. Positive control data

were not reported.

Metric 6:

Assay Procedures

Not Rated

Assay methods and procedures were cited to other

publications.

Metric 7:

Standards for Tests

Not Rated

This metric is not applicable to this study type.

Domain 3: Exposure Characterization

Metric 8:

Preparation and Storage of Test Substance

Not Rated

Assay methods were cited to other publications.

Metric 9:

Consistency of Exposure Administration

Not Rated

Assay methods were cited to other publications.

Metric 10:

Reporting of Doses/Concentrations

Not Rated

Assay methods were cited to other publications.

Metric 11:

Number of Exposure Groups and Concentra-

Not Rated

The assay procedures were described as "routine

tion Spacing

protocol" and cited in other references.

Metric 12:

Exposure Route and Method

Not Rated

The number of exposure groups and dose spacing

were not reported. The assay procedures were de-

scribed as "routine protocol" and cited in other ref-
erences

Metric 13:

Metabolic Activation

Medium

x 1

The source and method of preparation of the rat

liver S9 fraction was reported; however, the concen-

tration of S9 in the bacterial mutagenicity assay was

not specified.

Continued on next page . ..

Page 146 of 187

-------
.. . continued from previous page

Study Citation: V. V. Khudoley, I. Mizgireuv, G. B. Pliss (1987). The study of mutagenic activity of carcinogens and other chemical agents with

Salmonella typhimurium assays: Testing of 126 compounds Archiv fur Geschwulstforschung, 57(6,6), 453-462
Data Type: Bacterial reverse mutation for DCM

HERO ID: 194949

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 4: Test Model

Metric 14:

Test Model

High

x 2

The identity and donor source of the bacterial

strains used here were identified, and these strains
are routinely used for the outcome of interest.

Metric 15:

Number per Group

Not Rated

The number of plates per treatment group was not
reported. The assay procedures were described as
"routine protocol" and cited in other references.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

x 2

The outcome assessment methodology is appropriate
and senditive for the outcome of interest.

Metric 17:

Consistency of Outcome Assessment

High

x 1

The outcome assessment was consistent across treat-
ment groups.

Metric 18:

Sampling Adequacy

Not Rated

This metric is not applicable to this endpoint.

Metric 19:

Blinding of Assessors

Not Rated

Number of colonies is an objective outcome and
blinding assessors is not necessary.

Domain 6: Confounding / Variable Control

Metric 20:

Confounding Variables in Test Design and
Procedures

Low

x 2

Initial conditions were not reported for each study
replicate or group.

Metric 21:

Confounding Variables in Outcomes Unre-
lated to Exposure

Low

x 1

Data on outcome differences unrelated to exposure
were not reported for each study replicate or group.

Domain 7: Data Presentation and Analysis

Metric 22:

Data Analysis

Medium

x 1

Metric 23:

Data Interpretation

High

x 2

Evaluation criteria (number of colonies) was re-
ported and consistent with standards and guidelines.

Metric 24:

Cytotoxicity Data

Not Rated

No cytotoxicity assay was included for the bacterial
mutagenicity assay; however, this is unlikely to have
a substantial impact on the study results.

Metric 25:

Reporting of Data

High

x 2

All data are adequately reported.

Overall Quality Determination

Medium

1.7

Extracted

Yes

Continued on next page

Page 147 of 187

-------
. continued from previous page

Study Citation: V. V. Khudoley, I. Mizgireuv, G. B. Pliss (1987). The study of mutagenic activity of carcinogens and other chemical agents with

Salmonella typhimurium assays: Testing of 126 compounds Archiv fur Geschwulstforschung, 57(6,6), 453-462
Data Type:	Bacterial reverse mutation for DCM

HERO ID:	194949

Domain	Metric	Rating^ MWF* Score	Comments^

* MWF = Metric Weighting Factor

t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

+ The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

if any metric is Unacceptable

Overall rating =

J]. (Metric Score; x MWF;) / J] . MWFj

(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 148 of 187

-------
Table 51: In vitro evaluation results of Morita and Hayashi 1998 for sister chromatid exchange

Study Citation: T. Morita, M. Hayashi (1998). 1,4-Dioxane is not mutagenic in five in vitro assays and mouse peripheral blood micronucleus assay, but

is in mouse liver micronucleus assay Environmental and Molecular Mutagenesis, 32(3,3), 269-280
Data Type: Sister chromatid exchange

HERO ID: 195065

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

The test substance was identified as 1,4-dioxane with

the correct CASRN.

Metric 2:

Test Substance Source

High

The commercial source of the test substance was re-

ported.

Metric 3:

Test Substance Purity

High

The test substance was reported to be 99.8% pure.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

Appropriate concurrent negative control groups were
included (saline).

Metric 5:

Positive Controls

High

Appropriate concurrent positive control test sub-
stances were included with and without S9 acti-
vation (mitomycin C and benzo[a]pyrene, respec-
tively). Positive control groups exhibited positive
responses.

Metric 6:

Assay Procedures

High

Assay methods and procedures were described ade-
quately.

Metric 7:

Standards for Tests

Not Rated

This metric is not applicable to this study type.

Domain 3: Exposure Characterization

Metric 8:

Preparation and Storage of Test Substance

High

Test substance preparation was reported. Test sub-
stance storage was not reported (single-dose admin-
istration).

Metric 9:

Consistency of Exposure Administration

High

Exposure administration was consistent across treat-
ment groups.

Metric 10:

Reporting of Doses/Concentrations

High

The doses were reported without ambiguity.

Metric 11:

Number of Exposure Groups and Concentra-
tion Spacing

High

The exposure duration was reported and appropri-
ate.

Metric 12:

Exposure Route and Method

High

The number of exposure groups and dose spacing
were appropriate and within the range of previous
in vitro assays (provided in Table 1).

Metric 13:

Metabolic Activation

Medium

The source and method of preparation of the rat
liver S9 fraction was reported. The concentration
and exposure duration were appropriate. However,
the concentration of S9 used for this assay was not
specified.

Domain 4: Test Model

Continued on next page . ..

Page 149 of 187

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.. . continued from previous page

Study Citation: T. Morita, M. Hayashi (1998). 1,4-Dioxane is not mutagenic in five in vitro assays and mouse peripheral blood micronucleus assay, but

is in mouse liver micronucleus assay Environmental and Molecular Mutagenesis, 32(3,3), 269-280
Data Type: Sister chromatid exchange

HERO ID: 195065

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 14:

Test Model

High

The identity, commercial source, doubling time, and

karyotype features of the Chinese hamster ovary

(CHO-K1) were identified. This strain is routinely

used for the outcome of interest.

Metric 15:

Number per Group

Medium

Each experimental condition was completed in du-

plicate.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

The outcome assessment methodology is appropriate

for the outcome of interest.

Metric 17:

Consistency of Outcome Assessment

High

The outcome assessment was consistent across treat-

ment groups.

Metric 18:

Sampling Adequacy

High

The sampling was adequate at 100 well-spread

metaphases (50/replicate) per experimental condi-
tion.

Metric 19:

Blinding of Assessors

Low

The authors did not describe coding slides prior to

scoring

Domain 6: Confounding / Variable Control

Metric 20:

Confounding Variables in Test Design and

High

No differences among treatment group parameters

Procedures

were reported.

Metric 21:

Confounding Variables in Outcomes Unre-

High

No confounding variables were reported.

lated to Exposure

Domain 7: Data Presentation and Analysis

Metric 22:

Data Analysis

Medium

Data were analyzed by ANOVA followed by Stu-

dent's t-test. Student's t-test is not an appropri-

ate test given the variety of experimental conditions

(>2 groups). However, raw data is provided, which

would allow for independent statistical analysis.

Metric 23:

Data Interpretation

High

Evaluation criteria (number of sister chromatid ex-

changes per cell) is consistent with current stan-

dards.

Metric 24:

Cytotoxicity Data

High

The assay was completed in conjunction with a mea-

surement of cytotoxicity (trypan blue exclusion).

Metric 25:

Reporting of Data

High

All data are adequately reported and include a range

and standard deviation

Overall Quality Determination

High

1.1

Extracted

Yes

Continued on next page . ..

Page 150 of 187

-------
. continued from previous page

Study Citation: T. Morita, M. Hayashi (1998). 1,4-Dioxane is not mutagenic in five in vitro assays and mouse peripheral blood micronucleus assay, but

is in mouse liver micronucleus assay Environmental and Molecular Mutagenesis, 32(3,3), 269-280
Data Type:	Sister chromatid exchange

HERO ID:	195065

Domain	Metric	Rating^ MWF* Score	Comments^

* MWF = Metric Weighting Factor

t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

+ The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

if any metric is Unacceptable

Overall rating =

J]. (Metric Score; x MWF;) / J] . MWFj

(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 151 of 187

-------
Table 52: In vitro evaluation results of Morita and Hayashi 1998 for in vitro micronucleus study

Study Citation: T. Morita, M. Hayashi (1998). 1,4-Dioxane is not mutagenic in five in vitro assays and mouse peripheral blood micronucleus assay, but

is in mouse liver micronucleus assay Environmental and Molecular Mutagenesis, 32(3,3), 269-280
Data Type: In vitro micronucleus

HERO ID: 195065

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

The test substance was identified as 1,4-dioxane with

the correct CASRN.

Metric 2:

Test Substance Source

High

The commercial source of the test substance was re-

ported.

Metric 3:

Test Substance Purity

High

The test substance was reported to be 99.8% pure.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

Appropriate concurrent negative control groups were
included (saline).

Metric 5:

Positive Controls

High

Appropriate concurrent positive control test sub-
stances were included with and without S9 acti-
vation (mitomycin C and benzo[a]pyrene, respec-
tively).

Metric 6:

Assay Procedures

High

Assay methods and procedures were described ade-
quately.

Metric 7:

Standards for Tests

Not Rated

This metric is not applicable to this study type.

Domain 3: Exposure Characterization

Metric 8:

Preparation and Storage of Test Substance

High

Test substance preparation was reported. Test sub-
stance storage was not reported (single-dose admin-
istration).

Metric 9:

Consistency of Exposure Administration

High

Exposure administration was consistent across treat-
ment groups.

Metric 10:

Reporting of Doses/Concentrations

High

The doses were reported without ambiguity.

Metric 11:

Number of Exposure Groups and Concentra-
tion Spacing

High

The exposure duration was reported and appropri-
ate.

Metric 12:

Exposure Route and Method

High

The number of exposure groups and dose spacing
were appropriate and within the range of previous
in vitro assays (provided in Table 1).

Metric 13:

Metabolic Activation

Medium

The source and method of preparation of the rat
liver S9 fraction was reported; however, the concen-
tration of S9 used for this assay was not specified.

Domain 4: Test Model

Continued on next page . ..

Page 152 of 187

-------
.. . continued from previous page

Study Citation: T. Morita, M. Hayashi (1998). 1,4-Dioxane is not mutagenic in five in vitro assays and mouse peripheral blood micronucleus assay, but

is in mouse liver micronucleus assay Environmental and Molecular Mutagenesis, 32(3,3), 269-280
Data Type: In vitro micronucleus

HERO ID: 195065

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 14:

Test Model

High

The identity, commercial source, doubling time, and

karyotype features of the Chinese hamster ovary

(CHO-K1) were identified. This strain is routinely

used for the outcome of interest.

Metric 15:

Number per Group

Medium

Each experimental condition was completed in du-

plicate.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

The outcome assessment methodology is appropriate

for the outcome of interest.

Metric 17:

Consistency of Outcome Assessment

High

The outcome assessment was consistent across treat-

ment groups.

Metric 18:

Sampling Adequacy

High

The sampling was adequate at 2,000 intact inter-

phase cells per experimental condition.

Metric 19:

Blinding of Assessors

Low

Authors do not describe coding slides prior to char-

acterization of micronucleus frequencies (as recom-

mended in OECD test guidelines)

Domain 6: Confounding / Variable Control

Metric 20:

Confounding Variables in Test Design and

High

No differences among treatment group parameters

Procedures

were reported.

Metric 21:

Confounding Variables in Outcomes Unre-

High

No confounding variables were reported.

lated to Exposure

Domain 7: Data Presentation and Analysis

Metric 22:

Data Analysis

Medium

Statistical analysis was not conducted. A positive

result was defined as 3x the solvent control value.

Raw data were provided that would enable an inde-

pendent statistical analysis.

Metric 23:

Data Interpretation

High

Evaluation criteria (percentage of cells with mi-

cronuclei) was consistent with standards and guide-

Metric 24:

Cytotoxicity Data

High

The micronucleus assay was completed in conjunc-

tion with a measurement of cytotoxicity (trypan blue

exclusion).

Metric 25:

Reporting of Data

Low

Data are reported as percent cells with micronu-

clei, averaging across duplicates (rather than pro-

viding information about variability across individ-

ual plates)

Overall Quality Determination1" High 1.3

Continued on next page . ..

Page 153 of 187

-------
. continued from previous page

Study Citation: T. Morita, M. Hayashi (1998). 1,4-Dioxane is not mutagenic in five in vitro assays and mouse peripheral blood micronucleus assay, but

is in mouse liver micronucleus assay Environmental and Molecular Mutagenesis, 32(3,3), 269-280
Data Type:	In vitro micronucleus

HERO ID:	195065

Domain

Metric

Rating^

MWF* Score

Comments^

Extracted



Yes







MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; x MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 154 of 187

-------
Table 53: In vitro evaluation results of Morita and Hayashi 1998 for mouse liver micronucleus assay mouse lymphoma tk assay
(MLA)

Study Citation: T. Morita, M. Hayashi (1998). 1,4-Dioxane is not mutagenic in five in vitro assays and mouse peripheral blood micronucleus assay, but

is in mouse liver micronucleus assay Environmental and Molecular Mutagenesis, 32(3,3), 269-280
Data Type: Mouse lymphoma tk assay (MLA)

HERO ID: 195065

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

The test substance was identified as 1,4-dioxane with

the correct CASRN.

Metric 2:

Test Substance Source

High

The commercial source of the test substance was re-

ported.

Metric 3:

Test Substance Purity

High

The test substance was reported to be 99.8% pure.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

Appropriate concurrent negative control groups were
included (saline).

Metric 5:

Positive Controls

High

Appropriate concurrent positive control test sub-
stances were included with and without S9 ac-
tivation (methyl methanesulfonate and cyclophos-
phamide). Positive control groups exhibited positive
responses.

Metric 6:

Assay Procedures

High

Assay methods and procedures were described ade-
quately.

Metric 7:

Standards for Tests

Not Rated

This metric is not applicable to this study type.

Domain 3: Exposure Characterization

Metric 8:

Preparation and Storage of Test Substance

High

Test substance preparation was reported. Test sub-
stance storage was not reported (single-dose admin-
istration).

Metric 9:

Consistency of Exposure Administration

High

Exposure administration was consistent across treat-
ment groups.

Metric 10:

Reporting of Doses/Concentrations

High

The doses were reported without ambiguity.

Metric 11:

Number of Exposure Groups and Concentra-
tion Spacing

High

The exposure duration was reported and appropri-
ate.

Metric 12:

Exposure Route and Method

High

The number of exposure groups and dose spacing
were appropriate and within the range of previous
in vitro assays (provided in Table 1).

Metric 13:

Metabolic Activation

High

The source, method of preparation, and concentra-

tion of the rat liver S9 fraction was reported.

Domain 4: Test Model

Continued on next page . ..

Page 155 of 187

-------
... continued from previous page

Study Citation: T. Morita, M. Hayashi (1998). 1,4-Dioxane is not mutagenic in five in vitro assays and mouse peripheral blood micronucleus assay, but

is in mouse liver micronucleus assay Environmental and Molecular Mutagenesis, 32(3,3), 269-280
Data Type: Mouse lymphoma tk assay (MLA)

HERO ID: 195065

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 14:

Test Model

High

x 2

The identity, donor source, and doubling time of the

mouse lymphoma cell line (L5178Y) were identified.

This strain is routinely used for the outcome of in-

Metric 15:

Number per Group

Medium

x 1

Each assay was plated in duplicate.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

x 2

The outcome assessment methodology is appropriate

for the outcome of interest.

Metric 17:

Consistency of Outcome Assessment

High

x 1

The outcome assessment was consistent across treat-

ment groups.

Metric 18:

Sampling Adequacy

Not Rated

This metric is not applicable to this study type.

Metric 19:

Blinding of Assessors

Not Rated

Number of colonies is an objective outcome and

blinding assessors is not necessary.

Domain 6: Confounding / Variable Control

Metric 20:

Confounding Variables in Test Design and

High

x 2

No differences among treatment group parameters

Procedures

were reported.

Metric 21:

Confounding Variables in Outcomes Unre-

High

x 1

No confounding variables were reported.

lated to Exposure

Domain 7: Data Presentation and Analysis

Metric 22:

Data Analysis

High

x 1

The data were appropriately analyzed by pairwise

comparison and linear trend tests.

Metric 23:

Data Interpretation

High

x 2

Evaluation criteria (number of colonies) was consis-

tent with standards and guidelines.

Metric 24:

Cytotoxicity Data

High

x 1

The mouse lymphoma assay standard protocol in-

cludes a measurement to account for cytotoxicity

(relative survival without selection agent).

Metric 25:

Reporting of Data

High

x 2

All data are adequately reported.

Overall Quality Determination

High

1.0

Extracted

Yes

MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; x MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

^ This metric met the criteria for high confidence as expected for this type of study

Page 156 of 187

-------
Table 54: In vitro evaluation results of Morita and Hayashi 1998 for chromosomal aberration study

Study Citation: T. Morita, M. Hayashi (1998). 1,4-Dioxane is not mutagenic in five in vitro assays and mouse peripheral blood micronucleus assay, but

is in mouse liver micronucleus assay Environmental and Molecular Mutagenesis, 32(3,3), 269-280
Data Type: Chromosomal aberration

HERO ID: 195065

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

The test substance was identified as 1,4-dioxane with

the correct CASRN.

Metric 2:

Test Substance Source

High

The commercial source of the test substance was re-

ported.

Metric 3:

Test Substance Purity

High

The test substance was reported to be 99.8% pure.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

Appropriate concurrent negative control groups were
included (water).

Metric 5:

Positive Controls

High

Metric 6:

Assay Procedures

High

Assay methods and procedures were described ade-
quately.

Metric 7:

Standards for Tests

Not Rated

This metric is not applicable to this study type.

Domain 3: Exposure Characterization

Metric 8:

Preparation and Storage of Test Substance

High

Test substance preparation was reported. Test sub-
stance storage was not reported (single-dose admin-
istration).

Metric 9:

Consistency of Exposure Administration

High

Exposure administration was consistent across treat-
ment groups.

Metric 10:

Reporting of Doses/Concentrations

High

The doses were reported without ambiguity.

Metric 11:

Number of Exposure Groups and Concentra-
tion Spacing

High

The exposure duration was reported and appropri-
ate.

Metric 12:

Exposure Route and Method

High

The number of exposure groups and dose spacing
were appropriate and within the range of previous
in vitro assays (provided in Table 1).

Metric 13:

Metabolic Activation

High

The source, method of preparation, and concentra-

tion of the rat liver S9 fraction was reported.

Domain 4: Test Model

Continued on next page . ..

Page 157 of 187

-------
.. . continued from previous page

Study Citation: T. Morita, M. Hayashi (1998). 1,4-Dioxane is not mutagenic in five in vitro assays and mouse peripheral blood micronucleus assay, but

is in mouse liver micronucleus assay Environmental and Molecular Mutagenesis, 32(3,3), 269-280
Data Type: Chromosomal aberration

HERO ID: 195065

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 14:

Test Model

High

The identity, commercial source, doubling time, and

karyotype features of the Chinese hamster ovary
(CHO-K1) were identified. This strain is routinely
used for the outcome of interest.

Metric 15:

Number per Group

Medium

Each experimental condition was completed in du-
plicate.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

The outcome assessment methodology is appropriate
for the outcome of interest.

Metric 17:

Consistency of Outcome Assessment

High

The outcome assessment was consistent across treat-
ment groups.

Metric 18:

Sampling Adequacy

Medium

The sampling was somewhat lacking at 200 well-
spread metaphases per experimental condition

Metric 19:

Blinding of Assessors

Low

The authors do not describe coding slides prior to

scoring chromosomal aberrations

Domain 6: Confounding / Variable Control

Metric 20:

Confounding Variables in Test Design and
Procedures

High

No differences among treatment group parameters
were reported.

Metric 21:

Confounding Variables in Outcomes Unre-
lated to Exposure

High

No confounding variables were reported.

Domain 7: Data Presentation and Analysis

Metric 22:

Data Analysis

High

The data were appropriately analyzed by Fisher's
exact test.

Metric 23:

Data Interpretation

High

Evaluation criteria (percentage of cells with chromo-
somal aberrations) was reported and consistent with
standards and guidelines.

Metric 24:

Cytotoxicity Data

High

The chromosomal aberration assay was completed
in conjunction with a measurement of cytotoxicity
(trypan blue exclusion).

Metric 25:

Reporting of Data

Medium

Data are reported in terms of aberrations per 100
cells (averaging across duplicate cultures) without
any information on variability between the two du-
plicates

Overall Quality Determination

High

1.2

Extracted

Yes

Continued on next page . ..

Page 158 of 187

-------
. continued from previous page

Study Citation: T. Morita, M. Hayashi (1998). 1,4-Dioxane is not mutagenic in five in vitro assays and mouse peripheral blood micronucleus assay, but

is in mouse liver micronucleus assay Environmental and Molecular Mutagenesis, 32(3,3), 269-280
Data Type:	Chromosomal aberration

HERO ID:	195065

Domain	Metric	Rating^ MWF* Score	Comments^

* MWF = Metric Weighting Factor

t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

+ The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

if any metric is Unacceptable

Overall rating =

J]. (Metric Score; x MWF;) / J] . MWFj

(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 159 of 187

-------
Table 55: In vitro evaluation results of Morita and Hayashi 1998 for bacterial reverse mutation study

Study Citation: T. Morita, M. Hayashi (1998). 1,4-Dioxane is not mutagenic in five in vitro assays and mouse peripheral blood micronucleus assay, but

is in mouse liver micronucleus assay Environmental and Molecular Mutagenesis, 32(3,3), 269-280
Data Type: Bacterial reverse mutation

HERO ID: 195065

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

The test substance was identified as 1,4-dioxane with

the correct CASRN.

Metric 2:

Test Substance Source

High

The commercial source of the test substance was re-

ported.

Metric 3:

Test Substance Purity

High

The test substance was reported to be 99.8% pure.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

Appropriate concurrent negative control groups were
included (water).

Metric 5:

Positive Controls

High

Appropriate concurrent positive control test sub-
stances were included for each S. typhimurium and
E. coli strain with and without S9 activation. Posi-
tive control groups exhibited positive responses.

Metric 6:

Assay Procedures

High

Assay methods and procedures were described ade-
quately.

Metric 7:

Standards for Tests

Not Rated

This metric is not applicable to this study type.

Domain 3: Exposure Characterization

Metric 8:

Preparation and Storage of Test Substance

High

Test substance preparation was reported. Test sub-
stance storage was not reported (single-dose admin-
istration).

Metric 9:

Consistency of Exposure Administration

High

Exposure administration was consistent across treat-
ment groups.

Metric 10:

Reporting of Doses/Concentrations

High

The doses were reported without ambiguity.

Metric 11:

Number of Exposure Groups and Concentra-
tion Spacing

High

The exposure duration was reported and appropri-
ate.

Metric 12:

Exposure Route and Method

High

The number of exposure groups and dose spacing
was within the range of previous in vitro assays (pro-
vided in Table 1) and additionally exceeded previ-
ous studies' dose ranges by an order of magnitude to
confirm lack of mutagenicity.

Metric 13:

Metabolic Activation

Medium

The source and method of preparation of the rat
liver S9 fraction was reported; however, the concen-
tration of S9 in the bacterial mutagenicity assay was

not specified.

Domain 4: Test Model

Continued on next page . ..

Page 160 of 187

-------
... continued from previous page

Study Citation: T. Morita, M. Hayashi (1998). 1,4-Dioxane is not mutagenic in five in vitro assays and mouse peripheral blood micronucleus assay, but

is in mouse liver micronucleus assay Environmental and Molecular Mutagenesis, 32(3,3), 269-280
Data Type: Bacterial reverse mutation

HERO ID: 195065

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 14:

Test Model

High

x 2

The identity and donor source of the bacterial

strains used here were identified, and these strains

are routinely used for the outcome of interest.

Metric 15:

Number per Group

High

x 1

Each assay was plated in triplicate.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

x 2

The outcome assessment methodology is appropriate

for the outcome of interest.

Metric 17:

Consistency of Outcome Assessment

High

x 1

The outcome assessment was consistent across treat-

ment groups.

Metric 18:

Sampling Adequacy

Not Rated

This metric is not applicable to this endpoint.

Metric 19:

Blinding of Assessors

Not Rated

Number of colonies is an objective outcome and

blinding assessors is not necessary.

Domain 6: Confounding / Variable Control

Metric 20:

Confounding Variables in Test Design and

High

x 2

No differences among treatment group parameters

Procedures

were reported.

Metric 21:

Confounding Variables in Outcomes Unre-

High

x 1

No confounding variables were reported.

lated to Exposure

Domain 7: Data Presentation and Analysis

Metric 22:

Data Analysis

High

x 1

The data were appropriately analyzed by Dunnett's

Metric 23:

Data Interpretation

High

x 2

Evaluation criteria (number of colonies) was re-

ported and consistent with standards and guidelines

Metric 24:

Cytotoxicity Data

Medium

x 1

No cytotoxicity assay was included for the bacterial

mutagenicity assay; however, this is unlikely to have

a substantial impact on the study results.

Metric 25:

Reporting of Data

High

x 2

All data are adequately reported.

Overall Quality Determination

High

1.1

Extracted

Yes

MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; x MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

^ This metric met the criteria for high confidence as expected for this type of study

Page 161 of 187

-------
Table 56: Animal toxicity evaluation results of Goldsworthy et al 1991 for gavage study in rats on hepatocyte DNA repair

HERO ID: 62925

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Test substance identified as "1,4-dioxane".

Metric 2:

Test Substance Source

High

The source of the test substance was reported. The

batch/lot number was not reported, but the test sub-
stance is not expected to vary in composition.

Metric 3:

Test Substance Purity

High

Test substance was reported to be of HPLC grade,
99.9% purity.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

Concurrent negative controls were utilized (water
and corn oil).

Metric 5:

Positive Controls

High

Two positive control groups were included in
this study (2-Acetylaminofluorene in corn oil and
dimethylnitrosamine in water).

Metric 6:

Randomized Allocation

Low

The study did not report how animals were allocated
to study groups.

Domain 3: Exposure Characterization

Metric 7:

Preparation and Storage of Test Substance

Medium

Preparation and storage of the test substance was
not reported, but test substance administered as sin-
gle gavage dose.

Metric 8:

Consistency of Exposure Administration

High

Consistent gavage volumes were administered.

Metric 9:

Reporting of Doses/Concentrations

High

Gavage doses were reported.

Metric 10:

Exposure Frequency and Duration

High

The exposure frequency and duration was reported
(single dose) and appropriate for this endpoint.

Metric 11:

Number of Exposure Groups and Dose Spac-
ing

High

A single dose level (the highest dose recommended
for this specific assay) was utilized and considered

adequate for evaluating changes in DNA repair ac-
tivity compared to controls.

Metric 12:

Exposure Route and Method

High

The route of exposure was appropriate for this end-
point.

Domain 4: Test Organism

Metric 13:

Test Animal Characteristics

High

Test animal characteristics were reported.

Metric 14:

Adequacy and Consistency of Animal Hus-
bandry Conditions

High

Animal husbandry conditions were adequate and
consistent across control and exposed groups.

Continued on

next page .

Page 162 of 187

-------
... continued from previous page

Study Citation: T. L. Goldsworthy, T. M. Monticello, K. T. Morgan, E. Bermudez, D. M. Wilson, R. Jackh,Butterworth BE (1991). Examination of
potential mechanisms of carcinogenicity of 1,4-dioxane in rat nasal epithelial cells and hepatocytes Archives of Toxicology, 65(1,1), 1-9
Data Type:	Gavage Study - Hepatocyte DNA Repair

HERO ID:	62925

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 15:

Number per Group

High

X

1

1

The number of animals per treatment group was ad-
equate and appropriate for this endpoint (n = 3).

Domain 5: Outcome Assessment











Metric 16:

Outcome Assessment Methodology

High

X

2

2

The outcome assessment methodology was appropri-
ate for this endpoint.

Metric 17:

Consistency of Outcome Assessment

Medium

X

1

2

The study does not describe the timing of the out-











come assessment, but the same protocol was applied
for all groups.

Metric 18:

Sampling Adequacy

Medium

X

1

2

An adequate number of slides (n = 3) for each animal
was evaluated. However, the number of cells counted
for each slide (n = 25) is below what is required by
the OECD guideline (n = 100). The study authors
did not provide rationale for this difference, but cite
a different standard protocol.

Metric 19:

Blinding of Assessors

Not Rated

NA

NA

The outcome assessment relied on quantitative au-













toradiography. Blinding is not a concern in this
study.

Metric 20:

Negative Control Response

High

X

1

1

The control response was adequate.

Domain 6: Confounding / Variable Control











Metric 21:

Confounding Variables in Test Design and
Procedures

Medium

X

2

4

The study did not report on initial body weights
or food/water intake during this particular study,
but this is not likely to have a significant impact on
results.

Metric 22:

Health Outcomes Unrelated to Exposure

High

X

1

1

No health outcomes or deaths were reported in the











study.

Domain 7: Data Presentation and Analysis











Metric 23:

Statistical Methods

High

X

1

1

Statistical methods were reported and appropriate
for the dataset.

Metric 24:

Reporting of Data

High

X

2

2

Data were reported for all outcomes and groups.

Overall Quality Determination"



High





1.2



Extracted



Yes









MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; x MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 163 of 187

-------
Table 57: In vitro evaluation results of Munoz et al 2002 (195066) for a meiotic non-disjunction in Drosophila study

Study Citation: Munoz, ER; Barnett, BM (2002). The rodent carcinogens 1,4-dioxane and thiourea induce meiotic non-disjunction in Drosophila

melanogaster females Mutation Research, 517(1-2), 231-238
Data Type: Meiotic non-disjuntion in Drosophila

HERO ID: 195066

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1

Test Substance Identity

High

x 2

Test substance identified by name and CASRN.

Metric 2

Test Substance Source

Medium

x 1

Source identified by name.

Metric 3

Test Substance Purity

Low

x 1

Purity not reported.

Domain 2: Test Design

Metric 4

Negative and Vehicle Controls

High

x 2

Concurrent negative controls were included.

Metric 5

Positive Controls

Not Rated

Positive controls not required.

Metric 6

Assay Procedures

High

x 1

Assay procedures were described.

Metric 7

Standards for Tests

Not Rated

Criteria were not required.

Domain 3: Exposure Characterization

Metric 8

Preparation and Storage of Test Substance

Medium

x 1

Preparation of the test substance was briefly re-
ported and no storage information was reported.

Metric 9

Consistency of Exposure Administration

High

x 1

Exposures were administered consistently.

Metric 10: Reporting of Doses/Concentrations

High

x 2

The administered doses were reported.

Metric 11: Number of Exposure Groups and Concentra-

High

x 2

Exposure duration was reported.

tion Spacing

Metric 12: Exposure Route and Method

High

x 1

The number of groups and spacing were reported,
but justification was not reported.

Metric 13: Metabolic Activation

Not Rated

Metabolic activation was not required.

Domain 4: Test Model

Metric 14: Test Model

Medium

x 2

Test model and limited descriptive information were

reported.

Metric 15: Number per Group

High

x 1

The number of flies used was reported.

Domain 5: Outcome Assessment

Metric 16: Outcome Assessment Methodology

High

x 2

Outcome assessment methodology was reported.

Metric 17: Consistency of Outcome Assessment

High

x 1

Outcome assessment was consistent.

Metric 18: Sampling Adequacy

High

x 2

Sampling was adequate.

Metric 19: Blinding of Assessors

Not Rated

Blinding not required.

Domain 6: Confounding / Variable Control

Metric 20: Confounding Variables in Test Design and High x 2 2 There were no confounding variables in test design

Procedures anc^ Procedures.

Continued on next page . ..

Page 164 of 187

-------
... continued from previous page

Study Citation: Munoz, ER; Barnett, BM (2002). The rodent carcinogens 1,4-dioxane and thiourea induce meiotic non-disjunction in Drosophila

melanogaster females Mutation Research, 517(1-2), 231-238
Data Type:	Meiotic non-disjuntion in Drosophila

HERO ID:	195066

Domain Metric

Rating^

MWF*

Score

Comments^

Metric 21: Confounding Variables in Outcomes Unre-

High

X 1

1

No outcomes unrelated to exposure were reported.

lated to Exposure









Domain 7: Data Presentation and Analysis









Metric 22: Data Analysis

High

X 1

1

Appropriate analysis conducted.

Metric 23: Data Interpretation

Not Rated

NA

NA

Criteria not required.

Metric 24: Cytotoxicity Data

Not Rated

NA

NA

Data not required.

Metric 25: Reporting of Data

High

x 2

2

Data were reported.

Overall Quality Determination1"

High

1.2

Extracted

Yes





MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; x MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 165 of 187

-------
Table 58: In vitro evaluation results of Sheu et al 1988 for mammalian cell transformation

Study Citation: C. W. Sheu, F. M. Moreland, J. K. Lee, V. C. Dunkel (1988). In vitro BALB/3T3 cell transformation assay of nonoxynol-9 and

1,4-dioxane Environmental and Molecular Mutagenesis, 11(1,1), 41-48
Data Type: Mammalian cell transformation

HERO ID: 195078

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

x 2

The test substance was identified as 1,4-dioxane.

Metric 2:

Test Substance Source

High

x 1

The commercial source of the test substance was re-

ported, including manufacturer lot number.

Metric 3:

Test Substance Purity

Medium

x 1

The test substance was reported to be "certified ACS

grade," but purity was not reported.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

x 2

Appropriate concurrent negative control groups were
included (water).

Metric 5:

Positive Controls

High

x 2

Positive controls were tested concurrently with each

test substance. The identity of each positive control
was reported ("3-MCA", or methylcholanthrene)
and appropriate. Positive controls yielded positive
results.

Metric 6:

Assay Procedures

High

x 1

Assay methods and procedures were described in de-
tail and were applicable to the study type.

Metric 7:

Standards for Tests

Not Rated

This metric is not applicable to this study type.

Domain 3: Exposure Characterization

Metric 8:

Preparation and Storage of Test Substance

High

x 1

Test substance preparation was reported. Test sub-
stance storage was not reported; however, solutions
were prepared immediately before administration
(single-dose administration).

Metric 9:

Consistency of Exposure Administration

High

x 1

Exposure administration was consistent across treat-
ment groups.

Metric 10:

Reporting of Doses/Concentrations

High

x 2

The doses were reported without ambiguity.

Metric 11:

Number of Exposure Groups and Concentra-
tion Spacing

High

x 2

The exposure duration for the pre-incubation proto-
col was reported and appropriate.

Metric 12:

Exposure Route and Method

High

x 1

A dose level resulting in approximately 10% survival
in a preliminary cytotoxicity assay was selected as
the maximum dose in the transformation assay. The
number of exposure groups and dose spacing was
reported and appropriate for this assay (250, 500,
1000, or 2000 pg/mL).

Metric 13:

Metabolic Activation

Not Rated

This metric is not applicable to this study design.

Domain 4: Test Model

Continued on

next page . .

Page 166 of 187

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.. . continued from previous page

Study Citation: C. W. Sheu, F. M. Moreland, J. K. Lee, V. C. Dunkel (1988). In vitro BALB/3T3 cell transformation assay of nonoxynol-9 and

1,4-dioxane Environmental and Molecular Mutagenesis, 11(1,1), 41-48
Data Type: Mammalian cell transformation

HERO ID: 195078

Domain

Metric

Ratingt

MWF*

Score

Comments^

Metric 14:

Test Model

High

The identity, donor source, and passage number of
the cell line used here were identified, and this cell
line is appropriate for the outcome of interest.

Metric 15:

Number per Group

High

The experiment was conducted with 20 dishes per
treatment group.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

The outcome assessment methodology is appropriate
for the outcome of interest.

Metric 17:

Consistency of Outcome Assessment

High

The outcome assessment was consistent across treat-

ment groups.

Metric 18:

Sampling Adequacy

Not Rated

This metric is not applicable to this endpoint.

Metric 19:

Blinding of Assessors

High

It was reported that the identity of the dishes in
each group were coded.

Domain 6: Confounding / Variable Control

Metric 20:

Confounding Variables in
Procedures

Test Design and

High

No differences among treatment group parameters
were reported.

Metric 21:

Confounding Variables in
lated to Exposure

Outcomes Unre-

High

No confounding variables were reported.

Domain 7: Data Presentation and Analysis

Metric 22:

Data Analysis

Medium

The proportion of dishes with foci were analyzed us-
ing Fisher's exact test. The mean numbers of foci
per dish were analyzed using "a procedure described
by Lehmann (1959)." It was stated that the data
were assumed to have normal distributions, which
may not be a valid assumption.

Metric 23:

Data Interpretation

High

Evaluation criteria were reported and consistent
with current standards.

Metric 24:

Cytotoxicity Data

High

A dose-setting experiment was conducted to assess
cytotoxicity levels (viability, reduced numbers of
colonies). The doses for the mutagenicity assay were
selected so that the highest dose exhibited approxi-
mately 10% relative survival. Relative survival was
also assessed concurrently with the transformation
assay.

Metric 25:

Reporting of Data

High

All data are adequately reported.

Overall Quality Determination

High

1.1

Extracted

Yes

Continued on next page

Page 167 of 187

-------
. continued from previous page

Study Citation: C. W. Sheu, F. M. Moreland, J. K. Lee, V. C. Dunkel (1988). In vitro BALB/3T3 cell transformation assay of nonoxynol-9 and

1,4-dioxane Environmental and Molecular Mutagenesis, 11(1,1), 41-48
Data Type:	Mammalian cell transformation

HERO ID:	195078

Domain	Metric	Rating^ MWF* Score	Comments^

* MWF = Metric Weighting Factor

t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

+ The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

if any metric is Unacceptable

Overall rating =

J]. (Metric Score; x MWF;) / J] . MWFj

(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 168 of 187

-------
Table 59: In vitro evaluation report of Stott et al 1981 for bacterial reverse mutation study

Study Citation: W. T. Stott, J. F. Quast, P. G. Watanabe (1981). Differentiation of the mechanisms of oncogenicity of 1,4-dioxane and 1,3-

hexachlorobutadiene in the rat Toxicology and Applied Pharmacology, 60(2,2), 287-300
Data Type: Bacterial reverse mutation

HERO ID: 1937837

Domain

Metric

Rating^ MWF* Score

Comments^

Domain 1: Test Substance

Metric 1: Test Substance Identity

Metric 2: Test Substance Source

Metric 3: Test Substance Purity

High
High

High

X 2 2 1,4-Dioxane was identified by established nomencla-

ture.

X 1 1 The manufacturer of 1,4-dioxane was identified. A

lot number was not given; however, the material is
not expected to vary in composition.

X 1 1 The purity was reported as >99%.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

Metric 5: Positive Controls
Metric 6: Assay Procedures

Metric 7: Standards for Tests

High

High
Medium

x 2

x 2
x 1

Not Rated NA NA

Concurrent negative controls were used; however, it
was not clear whether cultures were untrated or ex-
posed to vehicle (saline).

Positive controls were used for each strain and pos-
itive responses were observed.

Methods were not fully described, but were de-
scribed as consistent with Ames, 1975 and appeared
to be appropriate.

This metric is not applicable to the outcomes of in-
terest.

Domain 3: Exposure Characterization

Metric 8: Preparation and Storage of Test Substance Medium x 1

Metric 9: Consistency of Exposure Administration High

Metric 10: Reporting of Doses/Concentrations High

Metric 11: Number of Exposure Groups and Concentra- Medium
tion Spacing

Metric 12: Exposure Route and Method
Metric 13: Metabolic Activation

High
High

x 1
x 2
x 2

x 1
x 1

2 The test substance was prepared in saline. Storage

conditions were not reported but this omission is un-
likely to have a substantial impact on results.

1 Exposures were administered consistently across
study groups .

2 Concentrations were reported without ambiguity (as
mg/ plate).

4 Method details are not provided; cited as Ames et
al. (1975) so duration is likely to be consistent with
assay standard.

1 5 concentration levels over 2 orders of magnitude.

1 The enzyme activating system (S9 mix) was a rat

liver homogenate obtained from Arochlor 1254~in-
duced animals.

Domain 4: Test Model

Continued on next page

Page 169 of 187

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.. . continued from previous page

Study Citation: W. T. Stott, J. F. Quast, P. G. Watanabe (1981). Differentiation of the mechanisms of oncogenicity of 1,4-dioxane and 1,3-

hexachlorobutadiene in the rat Toxicology and Applied Pharmacology, 60(2,2), 287-300
Data Type: Bacterial reverse mutation

HERO ID: 1937837

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 14:

Test Model

High

x 2

Source of the Salmonella test strains was listed as

TA 1535, 1537m 1538m 98, and 100.

Metric 15:

Number per Group

High

x 1

Data presented as mean of 3 replicates.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

x 2

The outcome assessment method addressed the in-

tended outcome(s) of interest (reverse mutation) and

was sensitive for the outcome(s) of interest.

Metric 17:

Consistency of Outcome Assessment

High

x 1

Outcomes were assessed consistently across groups.

Metric 18:

Sampling Adequacy

Not Rated

This metric was not applicable to the outcome of

interest.

Metric 19:

Blinding of Assessors

Not Rated

This metric was not applicable to the outcome of

interest.

Domain 6: Confounding / Variable Control

Metric 20:

Confounding Variables in Test Design and

High

x 2

No differences were reported among study groups.

Procedures

Metric 21:

Confounding Variables in Outcomes Unre-

High

x 1

No confounding variable unrelated to exposure were

lated to Exposure

identified.

Domain 7: Data Presentation and Analysis

Metric 22:

Data Analysis

Low

x 1

authors presented results as the mean across three

replicates + /- standard deviation; authors did not

describe methods for test significance of effects

across treatments, but sufficient data were provided

to conduct an independent statistical analysis.

Metric 23:

Data Interpretation

Not Rated

Data evaluation criteria cited to Ames et al. (1975).

Metric 24:

Cytotoxicity Data

Not Rated

This metric is not applicable to the outcome of in-

Metric 25:

Reporting of Data

High

x 2

Exposure-related findings were presented for all out-

comes by exposure group.

Overall Quality Determination

High

1.2

Extracted

Yes

Continued on next page . ..

Page 170 of 187

-------
. continued from previous page

Study Citation: W. T. Stott, J. F. Quast, P. G. Watanabe (1981). Differentiation of the mechanisms of oncogenicity of 1,4-dioxane and 1,3-

hexachlorobutadiene in the rat Toxicology and Applied Pharmacology, 60(2,2), 287-300
Data Type:	Bacterial reverse mutation

HERO ID:	1937837

Domain	Metric	Rating^ MWF* Score	Comments^

* MWF = Metric Weighting Factor

t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

+ The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

if any metric is Unacceptable

Overall rating =

J]. (Metric Score; x MWF;) / J] . MWFj

(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 171 of 187

-------
Table 60: In vitro evaluation results of Dow et al 1989 (4158028) for an unscheduled DNA synthesis-liver (p 248) study

Study Citation: Dow Chemical Company (1989). The evaluation of 1,3-hexachlorobutadiene and 1,4-dioxane in the rat hepatocyte unscheduled DNA
synthesis assay

Data Type: Unscheduled DNA synthesis-liver (p 248)

HERO ID: 4158028

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

x 2

Test substance identified by name, molecular weight

and formula, and structure.

Metric 2:

Test Substance Source

Medium

x 1

Source and lot number were reported.

Metric 3:

Test Substance Purity

Low

x 1

Purity not provided.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

x 2

Concurrent negative controls were include.

Metric 5:

Positive Controls

High

x 2

Concurrent positive control were included.

Metric 6:

Assay Procedures

High

x 1

Assay procedures were reported and were applicable
for the study type.

Metric 7:

Standards for Tests

Not Rated

This metric not applicable for the test.

Domain 3: Exposure Characterization

Metric 8:

Preparation and Storage of Test Substance

Medium

x 1

Test substance formulation was reported, but time
between preparation and use was not reported.

Metric 9:

Consistency of Exposure Administration

High

x 1

Exposures were administered consistently across
groups.

Metric 10:

Reporting of Doses/Concentrations

High

x 2

Concentrations were reported.

Metric 11:

Number of Exposure Groups and Concentra-
tion Spacing

High

x 2

Exposure duration was appropriate.

Metric 12:

Exposure Route and Method

High

x 1

The number of groups and concentration spaces were
adequate to address the purpose of the study, but
concentrations were not justified. .

Metric 13:

Metabolic Activation

Not Rated

Primary cultures do not have to be treated in the
presence and absence of metabolic activation.

Domain 4: Test Model

Metric 14:

Test Model

High

x 2

Test model strain, source, age, husbandry condi-
tions, and primary culture preparations were de-
scribed.

Metric 15:

Number per Group

High

x 1

The number of replicates (n=3) adequate for the
study type and outcome analysis.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

x 2

The outcome assessment methodology addressed the
intended outcome of interest.

Continued on

next page . .

Page 172 of 187

-------
. continued from previous page

Study Citation: Dow Chemical Company (1989). The evaluation of 1,3-hexachlorobutadiene and 1,4-dioxane in the rat hepatocyte unscheduled DNA
synthesis assay

Data Type:	Unscheduled DNA synthesis-liver (p 248)

HERO ID:	4158028

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 17:

Consistency of Outcome Assessment

High

X

1

1

Outcomes were assessed consistently.

Metric 18:

Sampling Adequacy

Medium

X

2

4

The number of cells/culture counted (n=15
cells/slide) were less than the minimum of 50
cells/culture recommended by OSCPP guideline
870.5550, but were sufficient for analyses.

Metric 19:

Blinding of Assessors

Medium

X

1

2

OSCPP guideline 870.5550 recommends coding











slides prior to counting cells, but it is not stated
that slides were coded before counting.

Domain 6: Confounding / Variable Control











Metric 20:

Confounding Variables in Test Design and
Procedures

High

X

2

2

There were no confounding variables in test design
and procedures.

Metric 21:

Confounding Variables in Outcomes Unre-
lated to Exposure

High

X

1

1

There were no reported differences among the repli-
cates or groups unrelated to exposure.

Domain 7: Data Presentation and Analysis











Metric 22:

Data Analysis

High

X

1

1

Statistical analyses were described and appropriate.

Metric 23:

Data Interpretation

High

X

2

2

The requirements for an unequivocal positive result
were reported.

Metric 24:

Cytotoxicity Data

High

X

1

1

The highest dose tested was cytotoxic which is the
only cytotoxicity recommendation for the assay.

Metric 25:

Reporting of Data

High

X

2

2

Data were presented for all outcomes.

Overall Quality Determination"



High





1.2



Extracted



Yes









MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; x MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 173 of 187

-------
Table 61: In vitro evaluation results of Dow et al 1989 (4158030) for a genotoxicity study in salmonella

Study Citation: Dow Chemical Company (1989). Differentiation of the mechanisms of oncogenicity of 1,4-dioxane and 1,3-hexachlorobutadiene in the
rat

Data Type: Genotoxicity-Salmonella (p. 262)

HERO ID: 4158030

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1

Test Substance Identity

Medium

x 2

Identified only by name.

Metric 2

Test Substance Source

Medium

x 1

Source only was identified.

Metric 3

Test Substance Purity

High

x 1

Purity such that any effects are the result of the test
substance.

Domain 2: Test Design

Metric 4

Negative and Vehicle Controls

High

x 2

Concurrent negative controls were used

Metric 5

Positive Controls

High

x 2

Concurrent positive controls were used

Metric 6

Assay Procedures

Medium

x 1

Specific details were not reported, however, the test

was conducted as described by Ames et al., 1975.

Metric 7

Standards for Tests

Not Rated

Not applicable for this test.

Domain 3: Exposure Characterization

Metric 8

Preparation and Storage of Test Substance

Medium

x 1

Formulation was reported, but time between prepa-
ration and use was not reported.

Metric 9

Consistency of Exposure Administration

Medium

x 1

Details of exposure administration were inferred

from the text as they were not stated for all con-
centrations groups.

Metric 10: Reporting of Doses/Concentrations
Metric 11: Number of Exposure Groups and Concentra-
tion Spacing
Metric 12: Exposure Route and Method

Metric 13: Metabolic Activation

High X 2 2 Concentrations were reported without ambiguity.

High X 2 2 Test conducted according to Ames et al. 1975-which

states —48-hour incubation.

Medium X 1 2 The number of groups and concentration spaces were

not justified, but were sufficient to address the pur-
pose of the study.

Medium X 1 2 The type and source of system were reported, but

some details (e.g., composition mix, volume in final
culture, concentration, QC information) were not in-
cluded.

Domain 4: Test Model

Metric 14: Test Model

Metric 15: Number per Group

Low
High

X 2 6 The test model was reported with no additional de-

tails.

X 1 1 Test conducted according to Ames et al. 1975-which

states to use 0.1 mL of culture.

Domain 5: Outcome Assessment

Continued on next page . ..

Page 174 of 187

-------
. continued from previous page

Study Citation: Dow Chemical Company (1989). Differentiation of the mechanisms of oncogenicity of 1,4-dioxane and 1,3-hexachlorobutadiene in the
rat

Data Type: Genotoxicity-Salmonella (p. 262)

HERO ID: 4158030

Domain

Metric

Rating^ MWF* Score

Comments^

Metric 16:

Outcome Assessment Methodology

Medium

Test conducted according to Ames et al. 1975-which

states to count the colonies and provides sponta-
neous revertant colony counts for east strain.

Metric 17:

Consistency of Outcome Assessment

High

There were no details reported or inferred that sug-
gested that outcome assessment was not consistent.

Metric 18:

Sampling Adequacy

High

Sampling was adequate for the outcome of interest.

Metric 19:

Blinding of Assessors

Not Rated

The metric is not applicable.

Domain 6: Confounding / Variable Control

Metric 20:

Confounding Variables in Test Design and
Procedures

High

There were no confounding variables in test design
or procedures.

Metric 21:

Confounding Variables in Outcomes Unre-
lated to Exposure

High

There were no reported differences among the repli-
cates unrelated to exposure.

Domain 7: Data Presentation and Analysis

Metric 22:

Data Analysis

High

No calculation methods were reported, but sufficient
data were provided to conduct independent analysis.

Metric 23:

Data Interpretation

Medium

The evaluation criteria were partially reported (i.e.,
increased in background reversion rate), but given
the results, the omission does not impact the results.

Metric 24:

Cytotoxicity Data

Medium

Cytotoxicity endpoints were partially defined (i.e.,
background lawn toxicity), but this is not detrimen-
tal to the results.

Metric 25:

Reporting of Data

High

Data for exposure-related findings were reported.

Overall Quality Determination"

High

1.5

Extracted

Yes

MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; x MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

Page 175 of 187

-------
Table 62: Animal toxicity evaluation results of Goldsworthy et al 1991 for drinking water study in rats on hepatocyte DNA repair

HERO ID: 62925

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Test substance identified as "1,4-dioxane".

Metric 2:

Test Substance Source

High

The source of the test substance was reported. The

batch/lot number was not reported, but the test sub-
stance is not expected to vary in composition.

Metric 3:

Test Substance Purity

High

Test substance was reported to be of HPLC grade,
99.9% purity.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

Concurrent negative controls were utilized (water
and corn oil).

Metric 5:

Positive Controls

High

Two positive control groups were included in
this study (2-Acetylaminofluorene in corn oil and
dimethylnitrosamine in water).

Metric 6:

Randomized Allocation

Low

The study did not report how animals were allocated
to study groups.

Domain 3: Exposure Characterization

Metric 7:

Preparation and Storage of Test Substance

Medium

Preparation and storage of the test substance was
not reported, but test substance administered in
drinking water and test substance is known to be
soluble in water.

Metric 8:

Consistency of Exposure Administration

High

Consistent concentration added to drinking water
provided to rats.

Metric 9:

Reporting of Doses/Concentrations

Low

Concentrations administered in drinking water were
reported. No palatability issues were described, but
body weights and water consumption were not re-
ported.

Metric 10:

Exposure Frequency and Duration

High

The exposure frequency and duration was reported
and appropriate for this endpoint.

Metric 11:

Number of Exposure Groups and Dose Spac-
ing

Medium

Number of exposure groups and spacing of exposure
levels were adequate to show results relevant to the

outcome of interest, but there was no justification
for why the doses and spacing were selected.

Metric 12:

Exposure Route and Method

High

The route of exposure was appropriate for this end-
point.

Domain 4: Test Organism

Metric 13:

Test Animal Characteristics

High

Test animal characteristics were reported.

Continued on

next page .

Page 176 of 187

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.. . continued from previous page

HERO ID: 62925

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 14:

Adequacy and Consistency of Animal Hus-
bandry Conditions

High

Animal husbandry conditions were adequate and
consistent across control and exposed groups.

Metric 15:

Number per Group

High

The number of animals per treatment group was ad-
equate and appropriate for this endpoint (n = 3).

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

The outcome assessment methodology was appropri-
ate for this endpoint.

Metric 17:

Consistency of Outcome Assessment

Medium

The study does not describe the timing of the out-

come assessment, but the same protocol was applied
for all groups.

Metric 18:

Sampling Adequacy

Medium

An adequate number of slides (n = 3) for each animal
was evaluated. However, the number of cells counted
for each slide (n = 25) is below what is required by
the OECD guideline (n = 100). The study authors
did not provide rationale for this difference, but cite
a different standard protocol.

Metric 19:

Blinding of Assessors

Not Rated

The outcome assessment relied on quantitative au-

toradiography. Blinding is not a concern in this
study.

Metric 20:

Negative Control Response

High

The control response was adequate.

Domain 6: Confounding / Variable Control

Metric 21:

Confounding Variables in Test Design and
Procedures

Medium

The study did not report on initial body weights or
food/water intake during this drinking water study.

Metric 22:

Health Outcomes Unrelated to Exposure

High

No health outcomes or deaths were reported in the

study.

Domain 7: Data Presentation and Analysis

Metric 23:

Statistical Methods

High

Statistical methods were reported and appropriate
for the dataset.

Metric 24:

Reporting of Data

High

Data were reported for all outcomes and groups.

Overall Quality Determination

High

1.4

Extracted

Yes

Continued on next page . ..

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-------
. continued from previous page

Study Citation: T. L. Goldsworthy, T. M. Monticello, K. T. Morgan, E. Bermudez, D. M. Wilson, R. Jackh,Butterworth BE (1991). Examination of
potential mechanisms of carcinogenicity of 1,4-dioxane in rat nasal epithelial cells and hepatocytes Archives of Toxicology, 65(1,1), 1-9
Data Type:	Drinking Water Study - Hepatocyte DNA Repair

HERO ID:	62925

Domain	Metric	Rating^ MWF* Score	Comments^

* MWF = Metric Weighting Factor

t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

+ The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

if any metric is Unacceptable

Overall rating =

J]. (Metric Score; x MWF;) / J] . MWFj

(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 178 of 187

-------
Table 63: Animal toxicity evaluation results of Goldsworthy et al 1991 for drinking water study in rats on hepatocyte cell proliferation

HERO ID: 62925

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Test substance identified as "1,4-dioxane".

Metric 2:

Test Substance Source

High

The source of the test substance was reported. The

batch/lot number was not reported, but the test sub-
stance is not expected to vary in composition.

Metric 3:

Test Substance Purity

High

Test substance was reported to be of HPLC grade,
99.9% purity.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

Concurrent negative controls were utilized (water).

Metric 5:

Positive Controls

Not Rated

No positive control group was needed for this study

type.

Metric 6:

Randomized Allocation

Low

The study did not report how animals were allocated
to study groups.

Domain 3: Exposure Characterization

Metric 7:

Preparation and Storage of Test Substance

Medium

The study notes that the test substance was admin-
istered in water and the test substance is known to
be soluble in water. Storage conditions were not re-
ported.

Metric 8:

Consistency of Exposure Administration

High

The study reports that animals were continuously
administered a consistent concentration in the drink-
ing water.

Metric 9:

Reporting of Doses/Concentrations

Low

Concentrations in drinking water were reported
along with the total average water intake for the ex-
posed animals and the control animals. No palata-
bility issues were described, but daily water intake
rates and body weights were not reported.

Metric 10:

Exposure Frequency and Duration

High

The exposure frequency and duration (single dose)
was reported and appropriate for this endpoint.

Metric 11:

Number of Exposure Groups and Dose Spac-
ing

High

A single dose level was utilized, but this was consid-
ered adequate for evaluating hepatocyte cell replica-
tion at different time points compared to controls.

Metric 12:

Exposure Route and Method

High

The route of exposure was appropriate for this end-
point.

Domain 4: Test Organism

Metric 13:

Test Animal Characteristics

High

Test animal characteristics were reported.

Continued on

next page . .

Page 179 of 187

-------
. continued from previous page

HERO ID: 62925

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 14:

Adequacy and Consistency of Animal Hus-
bandry Conditions

High

Animal husbandry conditions were adequate and
consistent across control and exposed groups.

Metric 15:

Number per Group

Medium

The number of animals in the exposed treatment
groups was adequate for the outcome analysis (n =
5), but a smaller number of animals was included in
the negative control group (n = 3) without reference
to a historical dataset.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

The outcome assessment methodology was appropri-
ate for this endpoint.

Metric 17:

Consistency of Outcome Assessment

High

The outcome assessment as consistent for all groups.

Metric 18:

Sampling Adequacy

High

The number of hepatocyte nuclei (n=2,000) from
each liver section was adequate for the outcome of
interest.

Metric 19:

Blinding of Assessors

Not Rated

The outcome assessment relied on quantitative au-

toradiography. Blinding is not a concern in this
study.

Metric 20:

Negative Control Response

High

The control response was adequate.

Domain 6: Confounding / Variable Control

Metric 21:

Confounding Variables in Test Design and
Procedures

Medium

The study did not report on initial body weights
or food/water intake for individual animals during
this particular study, but this is not likely to have a
significant impact on results.

Metric 22:

Health Outcomes Unrelated to Exposure

High

No health outcomes or deaths were reported in the

study.

Domain 7: Data Presentation and Analysis

Metric 23:

Statistical Methods

High

Statistical methods were reported and appropriate
for the dataset.

Metric 24:

Reporting of Data

High

Data were reported for all outcomes and groups.

Overall Quality Determination"1' High L3

Extracted Yes

Continued on next page . ..

Page 180 of 187

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. continued from previous page

Study Citation: T. L. Goldsworthy, T. M. Monticello, K. T. Morgan, E. Bermudez, D. M. Wilson, R. Jackh,Butterworth BE (1991). Examination of
potential mechanisms of carcinogenicity of 1,4-dioxane in rat nasal epithelial cells and hepatocytes Archives of Toxicology, 65(1,1), 1-9
Data Type:	Drinking Water Study - Hepatocyte Cell Proliferation

HERO ID:	62925

Domain	Metric	Rating^ MWF* Score	Comments^

* MWF = Metric Weighting Factor

t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

+ The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

if any metric is Unacceptable

Overall rating =

J]. (Metric Score; x MWF;) / J] . MWFj

(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 181 of 187

-------
Table 64: Animal toxicity evaluation results of Goldsworthy et al 1991 for nasal cell proliferation in rats

HERO ID: 62925

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Test substance identified as "1,4-dioxane".

Metric 2:

Test Substance Source

High

The source of the test substance was reported. The

batch/lot number was not reported, but the test sub-
stance is not expected to vary in composition.

Metric 3:

Test Substance Purity

High

Test substance was reported to be of HPLC grade,
99.9% purity.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

Concurrent negative controls were utilized. Materi-
als and methods does not specify a control group,
but footnote to Table 8 reports control responses.

Metric 5:

Positive Controls

Not Rated

No positive control group was needed for this study

type.

Metric 6:

Randomized Allocation

Low

The study did not report how animals were allocated
to study groups.

Domain 3: Exposure Characterization

Metric 7:

Preparation and Storage of Test Substance

Medium

The study notes that the test substance was admin-
istered in water and the test substance is known to
be soluble in water. Storage conditions were not re-
ported.

Metric 8:

Consistency of Exposure Administration

High

The study reports that animals were continuously
administered a consistent concentration in the drink-
ing water.

Metric 9:

Reporting of Doses/Concentrations

Low

Nominal concentration administered in drinking wa-
ter was reported, but actual doses were not reported.
No palatability issues were described, but water in-
take rates and body weights were not reported.

Metric 10:

Exposure Frequency and Duration

High

The exposure frequency and duration was reported
(single dose) and appropriate for this endpoint.

Metric 11:

Number of Exposure Groups and Dose Spac-
ing

High

A single dose level was utilized, but this was con-
sidered adequate for evaluating cell replication at
different time points compared to controls.

Metric 12:

Exposure Route and Method

High

The route of exposure was appropriate for this end-
point.

Domain 4: Test Organism

Metric 13:

Test Animal Characteristics

High

Test animal characteristics were reported.

Continued on

next page . .

Page 182 of 187

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. continued from previous page

HERO ID: 62925

Domain

Metric

Rating^

MWF*

Score

Comments^

Metric 14:

Adequacy and Consistency of Animal Hus-

High

X 1

Animal husbandry conditions were adequate and

bandry Conditions

consistent across control and exposed groups.

Metric 15:

Number per Group

Unacceptable

X 1

The number of animals/group was not reported.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

x 2

The outcome assessment methodology was appropri-

ate for this endpoint.

Metric 17:

Consistency of Outcome Assessment

High

x 1

The outcome assessment as consistent for all groups.

Metric 18:

Sampling Adequacy

High

x 1

Sampling adequacy was adequate for the specific

outcome of interest.

Metric 19:

Blinding of Assessors

Not Rated

Blinding is not a concern in this study.

Metric 20:

Negative Control Response

High

x 1

The control response was adequate.

Domain 6: Confounding / Variable Control

Metric 21:

Confounding Variables in Test Design and

Medium

x 2

The study did not report on initial body weights

Procedures

or food/water intake during this particular study,

but this is not likely to have a significant impact on

Metric 22:

Health Outcomes Unrelated to Exposure

High

x 1

No health outcomes or deaths were reported in the

study.

Domain 7: Data Presentation and Analysis

Metric 23:

Statistical Methods

High

x 1

Statistical methods were reported and appropriate

for the dataset.

Metric 24:

Reporting of Data

High

x 2

Data were reported for all outcomes and groups.

Overall Quality Determination'

Unacceptable**

1.4

Extracted

MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; X MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

^ This metric met the criteria for high confidence as expected for this type of study

Page 183 of 187

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7 Mechanistic

Table 65: In vitro evaluation results of Shah et al 2015 (3115011) for a hepatic CYP450 enzyme activity (metabolism) study

Study Citation: Shah, TS; Kamble, SH; Patil, PG; Iyer, KR (2015). Effect of water-miscible organic solvents on cyp450-mediated metoprolol and

imipramine metabolism in rat liver microsomes Indian Journal of Pharmaceutical Sciences, 77(4), 382-390
Data Type: Hepatic CYP450 enzyme activity (metabolism)

HERO ID: 3115011

Domain

Metric

Rating^

MWF*

Score

Comments^

Domain 1: Test Substance

Metric 1:

Test Substance Identity

Medium

x 2

Test substance identified by name only,

Metric 2:

Test Substance Source

Medium

x 1

Source identified by name only.

Metric 3:

Test Substance Purity

Low

x 1

Purity was not reported.

Domain 2: Test Design

Metric 4:

Negative and Vehicle Controls

High

x 2

Concurrent negative controls were exposed.

Metric 5:

Positive Controls

Not Rated

This metric not applicable.

Metric 6:

Assay Procedures

High

x 1

Assay procedures were described in detail and appli-

cable for the study type.

Metric 7:

Standards for Tests

Not Rated

This metric is not applicable.

Domain 3: Exposure Characterization

Metric 8:

Preparation and Storage of Test Substance

Medium

x 1

Formulation details were reported, but time between

preparation and use was not reported.

Metric 9:

Consistency of Exposure Administration

High

x 1

Exposures were administered consistently across

groups.

Metric 10:

Reporting of Doses/Concentrations

High

x 2

Concentrations and reaction volumes were reported

without ambiguity.

Metric 11:

Number of Exposure Groups and Concentra-

High

x 2

Exposure duration was reported and appropriate.

tion Spacing

Metric 12:

Exposure Route and Method

High

x 1

The number of groups and concentration spacing

were not justified by the study authors, but were

sufficient to address the purposes of the study.

Metric 13:

Metabolic Activation

Not Rated

This metric not applicable

Domain 4: Test Model

Metric 14:

Test Model

High

x 2

Microsomes were obtained from rats sacrificed

from other experiments, and were characterized for

CYP450 content.

Metric 15:

Number per Group

High

x 1

The number of replicates was reported and appro-

priate.

Domain 5: Outcome Assessment

Metric 16:

Outcome Assessment Methodology

High

x 2

Outcome assessment methodology was appropriate.

Metric 17:

Consistency of Outcome Assessment

High

x 1

Outcome assessment was conducted consistently.

Continued on

next page . .

Page 184 of 187

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. continued from previous page

Study Citation: Shah, TS; Kamble, SH; Patil, PG; Iyer, KR (2015). Effect of water-miscible organic solvents on cyp450-mediated metoprolol and

imipramine metabolism in rat liver microsomes Indian Journal of Pharmaceutical Sciences, 77(4), 382-390
Data Type:	Hepatic CYP450 enzyme activity (metabolism)

HERO ID:	3115011

Domain

Metric



Ratingt

MWF*

Score

Comments^

Metric 18:

Sampling Adequacy



High

x 2

2

Sampling was adequate.

Metric 19:

Blinding of Assessors



Not Rated

NA

NA

This metric is not applicable.

Domain 6: Confounding / Variable Control











Metric 20:

Confounding Variables in

Test Design and

Medium

x 2

4

The rats from which liver microsomes were collected



Procedures







were only described as having been part of other
experiments. It is unclear if the rats were obtained
from control groups or treated groups.

Metric 21:

Confounding Variables in
lated to Exposure

Outcomes Unre-

High

x 1

1

No reported differences among the study replicates
or groups were observed and the test substance did
not interfere with the assay.

Domain 7: Data Presentation and Analysis











Metric 22:

Data Analysis



High

x 1

1

Calculation methods were reported and appropriate
and sufficient data were provided to conduct statis-
tical analyses..

Metric 23:

Data Interpretation



Not Rated

NA

NA

This metric scored not applicable to this study type.

Metric 24:

Cytotoxicity Data



Not Rated

NA

NA

This metric not applicable

Metric 25:

Reporting of Data



High

x 2

2

Outcome data were reported in the text and in tab-
ular and graphical formats.

Overall Quality Determination





High



1.3



Extracted





Yes







MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; x MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

^ This metric met the criteria for high confidence as expected for this type of study

Page 185 of 187

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Table 66: In vitro evaluation results of Patil et al 2015 for a CYP2el activity in liver microsomes study

Study Citation: Patil, PG; Kamble, SH; Shah, TS; Iyer, KR (2015). Effect of water miscible organic solvents on p-nitrophenol hydroxylase (CYP2E1)

activity in rat liver microsomes Indian Journal of Pharmaceutical Sciences, 77(3), 283-289
Data Type: CYP2E1 activity in liver microsomes

HERO ID: 3117721

Domain Metric Rating^ MWF* Score Comments^

Domain 1: Test Substance

Metric 1

Test Substance Identity

Low

x 2

Test substance only identified by name.

Metric 2

Test Substance Source

Medium

x 1

Source identified only.

Metric 3

Test Substance Purity

Low

x 1

Purity not reported.

Domain 2: Test Design

Metric 4

Negative and Vehicle Controls

High

x 2

Concurrent negative controls were included.

Metric 5

Positive Controls

Not Rated

Positive control not applicable.

Metric 6

Assay Procedures

High

x 1

Assay procedures were described in detail.

Metric 7

Standards for Tests

Not Rated

This metric not applicable for this test.

Domain 3: Exposure Characterization

Metric 8

Preparation and Storage of Test Substance

Medium

x 1

Formulation protocol was included, but time be-
tween preparation and use was not reported.

Metric 9

Consistency of Exposure Administration

High

x 1

Exposures were administered consistently.

Metric 10: Reporting of Doses/Concentrations

High

x 2

Concentrations and reaction volume amounts were
well described.

Metric 11: Number of Exposure Groups and Concentra-

High

x 2

Exposure duration was reported and adequate.

tion Spacing

Metric 12: Exposure Route and Method

Medium

x 1

Concentrations were not justified, were appropriate
to address the purposes of the study.

Metric 13: Metabolic Activation

Not Rated

Metabolic activation not required for this assay.

Domain 4: Test Model

Metric 14: Test Model

High

x 2

Test model was described and reported to be char-

acterized for CYP450 content.

Metric 15: Number per Group

High

x 1

The number of replicates was adequate for outcome
analysis.

Domain 5: Outcome Assessment

Metric 16: Outcome Assessment Methodology

High

x 2

Outcome assessment methodology addressed the in-

tended outcome of interest.

Metric 17: Consistency of Outcome Assessment

High

x 1

Outcomes were assessed consistently across groups.

Metric 18: Sampling Adequacy

High

x 2

Sampling was adequate for outcomes of interest.

Metric 19: Blinding of Assessors

Not Rated

No subjective outcomes assessed.

Domain 6: Confounding / Variable Control

Continued on next page . ..

Page 186 of 187

-------
.. . continued from previous page

Study Citation: Patil, PG; Kamble, SH; Shah, TS; Iyer, KR (2015). Effect of water miscible organic solvents on p-nitrophenol hydroxylase (CYP2E1)

activity in rat liver microsomes Indian Journal of Pharmaceutical Sciences, 77(3), 283-289
Data Type:	CYP2E1 activity in liver microsomes

HERO ID:	3117721

Domain

Metric



Ratingt

MWF*

Score

Comments^

Metric 20:

Confounding Variables in

Test Design and

Medium

x 2

4

Livers from rats sacrificed from other experiments



Procedures









were used but no additional data on the rats (i.e.,











control or treated) were reported.

Metric 21:

Confounding Variables in

Outcomes Unre-

High

x 1

1

There were no reported differences among the repli-



lated to Exposure









cates unrelated to exposure, and the test substance











did not interfere with the assay,

Domain 7: Data Presentation and Analysis











Metric 22:

Data Analysis



High

x 1

1

Calculation methods were described and data were









reported in which statistical analyses can be con-













ducted.

Metric 23:

Data Interpretation



Not Rated

NA

NA

Data evaluation criteria not required for this test.

Metric 24:

Cytotoxicity Data



Not Rated

NA

NA

Cytotoxicity was not measured.

Metric 25:

Reporting of Data



High

x 2

2

Outcome data were reported in the text and in tab-













ular and graphical formats.

Overall Quality Determination"1





High



1.4



Extracted

Yes

MWF = Metric Weighting Factor

High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value.

The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High.

Overall rating =

]T\ (Metric Score; X MWF;) / J] . MWFj

if any metric is Unacceptable
(round to the nearest tenth) otherwise

where High => 1 to < 1.7; Medium => 1.7 to < 2.3; Low => 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is
crossed out and an arrow points to the new rating.

This metric met the criteria for high confidence as expected for this type of study

Page 187 of 187

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