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


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&EPA

United States

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

d

O

June 2019

1

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Table of Contents

1.	Acute Toxicity Studies	4

1.1.	Animal toxicity evaluation results of Drew et al 1978 for a 4-hour inhalation
study on clinical chemistry/biochemical outcomes (hepatic enzymes]	4

1.2.	Animal toxicity evaluation results of Mattie et al 2012 for a 6-hour inhalation
study - neuro study on neurological/behavioral outcomes	10

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

2.	Short-term Toxicity Tests	20

2.1.	Animal toxicity evaluation results of Giavini et al 1985 for a developmental-fetal
effects study on growth (early life] and development outcomes	20

2.2.	Animal toxicity evaluation results of Goldberg et al 1964 for a 10-day inhalation
study on neurological/behavior, body weight outcomes	23

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

2.4.	Animal toxicity evaluation results of Mattie et al 2012 for a 2-week inhalation
study - systemic effects study on hepatic, renal, irritation, respiratory, hematological
and clinical chemistry outcomes	34

3.	Subchronic and Chronic Toxicity Studies (Including Cancer]	39

3.1.	Animal toxicity evaluation results of Kano et al 2008 for a 13-week oral toxicity
of 1,4-d in rats and mice study	39

3.2.	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	44

3.3.	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

3.4.	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	54

3.5.	Animal toxicity evaluation results of Argus et al 1965 for a cancer bioassay-
liver, kidney, blood study on cancer outcomes	60

3.6.	Animal toxicity evaluation results of Argus et al 1973 for a carcinogenicity-liver
(dose response], electron microscopy study on cancer outcomes	63

3.7.	Animal toxicity evaluation results of Jbrc etal 1998 for a cancer bioassay and
non-neoplastic lesions study on cancer, renal, hepatic, respiratory outcomes	66

3.8.	Animal toxicity evaluation results of Kano et al 2009 for a 2-year cancer
bioassay study on cancer outcomes	69

3.9.	Animal toxicity evaluation results of Kasai et al 2009 for a 2-year cancer
bioassay study on cancer, mortality, hepatic, renal, respiratory, hematological and

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immune, clinical chemistry/biochemical, nutrition and metabolic/adult exposure
body weight, reproductive outcomes	71

3.10.	Animal toxicity evaluation results of NCI et al 1978 for a cancer bioassay-
male rats study on cancer outcomes	76

3.11.	Animal toxicity evaluation results of NCI et al 1978 for a cancer bioassay-
female rats and male and female mice study on cancer outcomes	85

4.	In Vivo and In Vitro Genotoxicity Studies	94

4.1.	Animal toxicity evaluation results of Dow et al 1989 for a single dose in vivo
DNA synthesis study on hepatic, genotoxicity, body weight outcomes	94

4.2.	Animal toxicity evaluation results of Itoh 2019 - in vivo genotoxicity assay -
micronucleus test	98

4.3.	Animal toxicity evaluation results of Uno et al 1994 for an acute oral study on
mechanistic (gene expression/omics, genotoxicity] outcomes	102

4.4	Animal toxicity evaluation results of Dow et al 1989 for a repeat dose in vivo
DNA synthesis study on hepatic, genotoxicity, body weight outcomes	106

4.5	Animal toxicity evaluation results of Roy et al 2 005 for an in vivo micronucleus
assay - main study on genotoxicity outcomes	110

4.6	Animal toxicity evaluation results of Roy et al 2005 for an in vivo micronucleus
assay - range-finding study on genotoxicity, mortality outcomes	113

4.7.	In vitro evaluation results of Dow etal 1989 (4158028] for an unscheduled
DNA synthesis-liver (p 248] study	117

4.8.	In vitro evaluation results of Dow et al 1989 (4158030] for a genotoxicity-
salmonella study	120

4.9.	In vitro evaluation results of Munoz etal 2002 (195066] for a meiotic non-
disjunction in Drosophila study	123

4.10.	In vitro evaluation results ofZimmermann etal 1985 (194343]	125

5.	In Vitro Hepatic Metabolism Studies	128

5.1. In vitro evaluation results of Shah etal 2015 (3115011] for a hepatic CYP450
enzyme activity (metabolism] study	128

6.2 In vitro evaluation results of Patil et al 2015 for a CYP2el activity in liver
microsomes study	131

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1. Acute Toxicity Studies

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

Study reference:

Drew, R. T.,Patel, J. M.,Lin, F. N. (1978). Changes in serum enzymes in rats after inhalation of organic solvents
singly and in combination Toxicology and Applied Pharmacology, 45(3), 809-819. HERO ID: 67913

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Test Substance

1. Test Substance
Identity

The test substance was
identified definitively (by
name).

High

2. Test Substance
Source

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.

Low

3. Test Substance
Purity

Test substance purity
was reported as >99%.

High

Test Design

4. Negative and
Vehicle Controls

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.

Low

5. Positive Controls

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

Not Rated

6. Randomized
Allocation

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

Low

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Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Exposure
Characterization

7. Preparation and
Storage of Test
Substance

The study did not
completely report the
method and equipment
used to generate the test
substance atmosphere;
however, there was no
reason to believe that
there was an impact on

animal exposure.
Information on storage

was not reported;
however, there was no
reason to suggest that
the test substance was
unstable.

Medium

8. Consistency of

Exposure
Administration

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

Medium

9. Reporting of

Doses/
Concentrations

Concentrations were
reported as nominal
values. Vapor test
concentrations were
monitored continuously
by an automatic gas
sampling gas
chromatograph;
however, actual
concentrations were not
reported. Due to the lack

of reporting of actual
concentrations for vapor
exposures, 1 downgraded
this metric to low.

Low

10. Exposure
Frequency and
Duration

Exposure duration and
frequency were reported
(4 hours, one exposure)

and suitable for the
study type and outcomes
of interest.

High

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Study reference:

Drew, R. T.,Patel, J. M.,Lin, F. N. (1978). Changes in serum enzymes in rats after inhalation of organic solvents
singly and in combination Toxicology and Applied Pharmacology, 45(3), 809-819. HERO ID: 67913

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score



11. Number of
Exposure Groups
and Dose Spacing

The number of exposure
groups and
concentration spacing
(1000 and 2000) ppm
were relevant for the
assessment.

High

1

1

1

12. Exposure Route
and Method

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 1
downgraded the score to
low.

Low

3

1

3

Test Organism

13. Test Animal
Characteristics

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.

Medium

2

2

4

14. Adequacy and

Consistency of
Animal Husbandry
Conditions

Husbandry conditions
(temperature, humidity,

light cycle) were not
sufficiently reported to
evaluate if husbandry
was adequate and
similar among the
groups, so 1 downgraded
the score for this metric
to low.

Low

3

1

3

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Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

The exact number of

animals per group was

not reported. The
authors stated that each
experiment started with
15 animals,, The authors
stated that consecutive
daily heart punctures,
which were performed
to collect blood for

15. Number per
Group

serum enzyme assay
analyses, resulted in
several deaths, but the
exact number of deaths,

or final number of
animals/blood samples
collected per group, was

not reported.
Nevertheless, the results
appear to have been
sufficient for statistical
analysis, so 1 scored this
metric as medium.

Medium

16. Outcome
Assessment
Methodology

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

Low

Outcome
Assessment

17. Consistency of
Outcome
Assessment

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

High

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Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

18. Sampling
Adequacy

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

High

19. Blinding of
Assessors

No subjective endpoints
were evaluated in this
study.

Not Rated

20. Negative Control
Response

Each rat served as its
own control prior to
exposure.

High

Confounding /
Variable Control

21. Confounding
Variables in Test
Design and
Procedures

There were no
confounding differences

reported among the
study groups; however,
initial body weight or
food/water intake were

not reported.
Additionally, respiratory
rate was not reported,
but 1,4-dioxane is a
potential respiratory
irritant, so 1 downgraded
the score to low.

Low

22. Health
Outcomes Unrelated
to Exposure

Data on attrition and

health outcomes
unrelated to exposure
for each study group
were not reported
because only differences
among groups for the
evaluated outcomes
were noted.

Medium

Data Presentation
and Analysis

23. Statistical
Methods

Statistical methods were
described in sufficient
detail and were
appropriate for the data
sets.

High

24. Reporting of
Data

Data presentation is
incomplete. No data
were presented for
control groups.

Low

Sum of scores:

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Study reference:

Drew, R. T.,Patel, J. M.,Lin, F. N. (1978). Changes in serum enzymes in rats after inhalation of organic solvents
singly and in combination Toxicology and Applied Pharmacology, 45(3), 809-819. HERO ID: 67913

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3

Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:

2.1724

Overall Score:
Nearest *:

2.2

Overall Quality Level:

Medium

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

Study reference:

Mattie, D. R.,Bucher, T. W.,Carter, A. L.,Stoffregen, D. E.,Reboulet, J. E. (2012). Acute Inhalation Toxicity Study of
1, 4-Dioxane in Rats (Rattus norvegicus) GRA and 1(20), 29. HERO ID: 3563367

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

1. Test Substance
Identity

Clearly identified: 1,4-

dioxane ((formula:
C4H802); CAS # 123-91-

High

Test Substance

2. Test Substance
Source

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

Medium

3. Test Substance
Purity

>99% purity

High

Test Design

4. Negative and
Vehicle Controls

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).

High

5. Positive Controls

Positive control not
required for study type
(OPPTS 870.1300)

Not Rated

6. Randomized
Allocation

Animals were "randomly
selected for each
exposure group".

High

7. Preparation and
Storage of Test
Substance

Vapor generation
method was adequateely
reported.

High

Exposure
Characterization

8. Consistency of

Exposure
Administration

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.

Medium

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Study reference:

Mattie, D. R.,Bucher, T. W.,Carter, A. L.,Stoffregen, D. E.,Reboulet, J. E. (2012). Acute Inhalation Toxicity Study of
1, 4-Dioxane in Rats (Rattus norvegicus) GRA and 1(20), 29. HERO ID: 3563367

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

9. Reporting of

Doses/
Concentrations

Target, nominal, and
analytical concentrations
reported (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.

High

10. Exposure
Frequency and
Duration

Exposure duration
consistent with cited
guideline (OPPTS
870.1300)

High

11. Number of
Exposure Groups
and Dose Spacing

Five exposure groups
plus concurrent controls
were used. Exposure
levels were based on
levels in previous
studies.

High

12. Exposure Route
and Method

Dynamic, whole-body
exposure with 15
complete fresh air
changes per hour;
individually housed in
690 L chambers. Any
aerosols that were

formed during
vaporization process
were captured by a
patch of glass wool
upstream, so nose-only
exposure was not
necessary.

High

Test Organism

13. Test Animal
Characteristics

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.

High

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Study reference:

Mattie, D. R.,Bucher, T. W.,Carter, A. L.,Stoffregen, D. E.,Reboulet, J. E. (2012). Acute Inhalation Toxicity Study of
1, 4-Dioxane in Rats (Rattus norvegicus) GRA and 1(20), 29. HERO ID: 3563367

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

14. Adequacy and

Consistency of
Animal Husbandry
Conditions

Husbandry conditions
were the same between

groups. All animals
acclimated to exposure
chambers for 5 days
before exposure.

High

15. Number per
Group

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)

High

16. Outcome
Assessment
Methodology

Clinical signs of
neurotoxicity (autonomic
effects, central nervous

system effects, and
reactivity to handling or
sensory stimuli)

High

17. Consistency of
Outcome
Assessment

Assessment identical
across groups.

High

Outcome
Assessment

18. Sampling
Adequacy

Sampling consisted with
cited guideline (OPPTS
870.1300)

High

19. Blinding of
Assessors

No reporting of blinding
status of examiners
during subjective
assessments of clinical
signs of neurotoxicity.

Unacceptable

20. Negative Control
Response

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

Unacceptable

Confounding /
Variable Control

21. Confounding
Variables in Test
Design and
Procedures

Methods section states
that evaluations of
respiration 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
respiratory irritant.

Medium

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Study reference:

Mattie, D. R.,Bucher, T. W.,Carter, A. L.,Stoffregen, D. E.,Reboulet, J. E. (2012). Acute Inhalation Toxicity Study of
1, 4-Dioxane in Rats (Rattus norvegicus) GRA and 1(20), 29. HERO ID: 3563367

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

22. Health
Outcomes Unrelated
to Exposure

No mortalities were
reported. Minimal
serous exudate and few

acute and chronic
leukocyte infiltrates that
were observed in a small
number of rats
distributed 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.

Medium

Data Presentation
and Analysis

23. Statistical
Methods

No mention of statistical

analysis of clinical
neurotoxicity evaluation
(data not reported).

Unacceptable

24. Reporting of
Data

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

Unacceptable

High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3

Sum of scores:

Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:

1.6667

Overall Score
(Rounded):

1.71

Overall Quality Level:

Unacceptable1

Footnote:

1 Consistent with our Application of Systematic Review in TSCA Risk 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, three of the metrics were rated as
unacceptable. As such, the study is considered unacceptable and the score is presented solely to increase transparency.

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

Study reference:

Mattie, D. R.,Bucher, T. W.,Carter, A. L.,Stoffregen, D. E.,Reboulet, J. E. (2012). Acute Inhalation Toxicity Study of
1, 4-Dioxane in Rats (Rattus norvegicus) GRAand 1(20), 29. HERO ID: 3563367

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

1. Test Substance
Identity

Clearly identified: 1,4-

dioxane ((formula:
C4H802); CAS # 123-91-

High

Test Substance

2. Test Substance
Source

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

Medium

3. Test Substance
Purity

>99% purity

High

Test Design

4. Negative and
Vehicle Controls

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).

High

5. Positive Controls

Positive control not
required for study type
(OPPTS 870.1300)

Not Rated

6. Randomized
Allocation

Animals were "randomly
selected for each
exposure group".

High

7. Preparation and
Storage of Test
Substance

Vapor generation
method was adequately
reported.

High

Exposure
Characterization

8. Consistency of

Exposure
Administration

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.

Medium

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Study reference:

Mattie, D. R.,Bucher, T. W.,Carter, A. L.,Stoffregen, D. E.,Reboulet, J. E. (2012). Acute Inhalation Toxicity Study of
1, 4-Dioxane in Rats (Rattus norvegicus) GRA and 1(20), 29. HERO ID: 3563367

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

9. Reporting of

Doses/
Concentrations

Target, nominal, and
analytical concentrations
reported (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.

High

10. Exposure
Frequency and
Duration

Exposure duration
consistent with cited
guideline (OPPTS
870.1300)

High

11. Number of
Exposure Groups
and Dose Spacing

Five exposure groups
plus concurrent controls
were used. Exposure
levels were based on
levels in previous
studies.

High

12. Exposure Route
and Method

Dynamic, whole-body
exposure with 15
complete fresh air
changes per hour;
individually housed in
690 L chambers. Any
aerosols that were

formed during
vaporization process
were captured by a
patch of glass wool
upstream, so nose-only
exposure was not
necessary.

High

Test Organism

13. Test Animal
Characteristics

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.

High

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Mattie, D. R.,Bucher, T. W.,Carter, A. L.,Stoffregen, D. E.,Reboulet, J. E. (2012). Acute Inhalation Toxicity Study of
1, 4-Dioxane in Rats (Rattus norvegicus) GRA and 1(20), 29. HERO ID: 3563367

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score



14. Adequacy and

Consistency of
Animal Husbandry
Conditions

Husbandry conditions
were the same between

groups. All animals
acclimated to exposure
chambers for 5 days
before exposure.

High

1

1

1



15. Number per
Group

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)

High

1

1

1



16. Outcome
Assessment
Methodology

Hepatic, Renal - OW, HP
Respiratory - HP of entire

respiratory tract,
including nasal sections

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

High

1

2

2

Outcome
Assessment

17. Consistency of
Outcome
Assessment

Assessment identical
across groups.

High

1

1

1



18. Sampling
Adequacy

Sampling consisted with
cited guideline (OPPTS
870.1300)

High

1

1

1



19. Blinding of
Assessors

Only non-subjective
outcomes and initial
histopathological
evaluations performed;
blinding not necessary.

Not Rated

NA

NA

NA

16

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Study reference:

Mattie, D. R.,Bucher, T. W.,Carter, A. L.,Stoffregen, D. E.,Reboulet, J. E. (2012). Acute Inhalation Toxicity Study of
1, 4-Dioxane in Rats (Rattus norvegicus) GRA and 1(20), 29. HERO ID: 3563367

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

20. Negative Control
Response

Control histopathological
data were not explicitly
stated, but based on
qualitative statements
regarding what was
found in higher exposure
groups, it is inferred that
lesions were not
observed in controls.
Qualitative statement
regarding no statistically
significant changes in
organ weight or body
weight covers both
control and exposure
groups.

Medium

Confounding /
Variable Control

21. Confounding
Variables in Test
Design and
Procedures

Low

22. Health
Outcomes Unrelated
to Exposure

No mortalities were
reported. Minimal
serous exudate and few

bacterial infection";
observed at both 2 day
and 14 day sacrifice.

High

-------
PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Mattie, D. R.,Bucher, T. W.,Carter, A. L.,Stoffregen, D. E.,Reboulet, J. E. (2012). Acute Inhalation Toxicity Study of
1, 4-Dioxane in Rats (Rattus norvegicus) GRA and 1(20), 29. HERO ID: 3563367

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Data Presentation
and Analysis

23. Statistical
Methods

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 incidence is 0/5
for groups without
explicitly reported
lesions, statistical
analysis could be
conducted . Incidental

findings that were
observed in "all groups"

were reported
qualitatively only (not
adequate for statistical
analysis).

Medium

2

1

2

24. Reporting of
Data

BW/OW - Qualitative (no

effects)

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

Medium

2

2

4

High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3

Sum of scores:



29

39

Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:

NA

Overall Score:
Nearest *:

NA

Overall Quality Level:

Medium

18

-------
PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Mattie, D. R.,Bucher, T. W.,Carter, A. L.,Stoffregen, D. E.,Reboulet, J. E. (2012). Acute Inhalation Toxicity Study of
1, 4-Dioxane in Rats (Rattus norvegicus) GRA and 1(20), 29. HERO ID: 3563367

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Study Quality
Comment:

The reviewer downgraded this study's overall quality rating. They noted: 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 from high. 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. Note: The original calculated score for this study was
1.4. This value is not presented above because the final rating was changed based on professional judgement.

19

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

2. Short-term Toxicity Tests

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

Study reference:

Giavini, E.,Vismara, C.,Broccia, M. L. (1985). Teratogenesis study of dioxane in rats Toxicology Letters, 26(1), 85-
88. HERO ID: 62924

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Test Substance

1. Test Substance
Identity

The test substance was
identified by name only

Low

2. Test Substance
Source

Source identified but no

other details were
reported. The omitted
details are unlikely to
have a substantial
impact on results.

Medium

3. Test Substance
Purity

Purity and impurity
identified; purity such
that effects due to test
substance.

High

Test Design

4. Negative and
Vehicle Controls

Appropriate controls
used.

High

5. Positive Controls

This metric is not
applicable.

Not Rated

6. Randomized
Allocation

The method of allocation
was not reported.

Low

Exposure
Characterization

7. Preparation and
Storage of Test
Substance

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

Medium

8. Consistency of

Exposure
Administration

Exposures administered
consistently

High

9. Reporting of

Doses/
Concentrations

Doses were reported
without ambiguity.

High

10. Exposure
Frequency and
Duration

Details were reported
and appropriate.

High

11. Number of
Exposure Groups
and Dose Spacing

Number of exposure
groups and spacing were
appropriate

High

12. Exposure Route
and Method

The route and method
were suited to the test
substance.

High

-------
PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Giavini, E.,Vismara, C.,Broccia, M. L. (1985). Teratogenesis study of dioxane in rats Toxicology Letters, 26(1), 85-
88. HERO ID: 62924

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Test Organism

13. Test Animal
Characteristics

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

Medium

14. Adequacy and

Consistency of
Animal Husbandry
Conditions

The humidity, light-dark
cycle,, temperature, and
availability of food and
water were reported.

The number of
animals/cage was not
reported.

Medium

15. Number per
Group

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

Medium

Outcome
Assessment

16. Outcome
Assessment
Methodology

Outcome assessment
methodology was
appropriate and
sensitive.

High

17. Consistency of
Outcome
Assessment

Outcomes were assessed
consistently.

High

18. Sampling
Adequacy

Sampling was adequate
for the outcomes of
interest.

High

19. Blinding of
Assessors

This metric was not
applicable.

Not Rated

20. Negative Control
Response

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

High

Confounding /
Variable Control

21. Confounding
Variables in Test
Design and
Procedures

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

Medium

-------
PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Giavini, E.,Vismara, C.,Broccia, M. L. (1985). Teratogenesis study of dioxane in rats Toxicology Letters, 26(1), 85-
88. HERO ID: 62924

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score



22. Health
Outcomes Unrelated
to Exposure

No health outcomes
unrelated to exposure
were reported or could
be inferred .

High

1

1

1

Data Presentation
and Analysis

23. Statistical
Methods

Statistical tests were

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

Medium

2

1

2

24. Reporting of
Data

Data were presented for
all outcomes by
exposure groups.

High

1

2

2

High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3

Sum of scores:



29

44

Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:

1.517

Overall Score:
Nearest *:

1.5

Overall Quality Level:

High

22

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

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

Study reference:

Goldberg, M. E.,Johnson, H. E.,Pozzani, U. C.,Smyth, H. F., Jr. (1964). Effect of repeated inhalation of vapors of
industrial solvents on animal behavior: 1. Evaluation of nine solvent vapors on pole-climb performance in rats
American Industrial Hygiene Association Journal, 25(4), 369-375. HERO ID: 58035

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Test Substance

1. Test Substance
Identity

Test substance was
identified definitively.

High

2. Test Substance
Source

The report states that
chemicals were obtained
commercially; 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.

Low

3. Test Substance
Purity

Purity and grade were
not reported.

Low

Test Design

4. Negative and
Vehicle Controls

A concurrent negative
control group was tested
and was appropriate.

High

5. Positive Controls

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

Not Rated

6. Randomized
Allocation

Animals were
randomized and
distributed into groups.

High

Exposure
Characterization

7. Preparation and
Storage of Test
Substance

Methods and equipment
used for generating the
test atmospheres were

reported; however,
storage conditions for
the test substance were

not reported, so 1
downgraded the score
for this metric to
medium.

Medium

8. Consistency of

Exposure
Administration

Details of the exposure

administration were
reported and exposures

were administered
consistently across study
groups.

High

-------
PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Goldberg, M. E.,Johnson, H. E.,Pozzani, U. C.,Smyth, H. F., Jr. (1964). Effect of repeated inhalation of vapors of
industrial solvents on animal behavior: 1. Evaluation of nine solvent vapors on pole-climb performance in rats
American Industrial Hygiene Association Journal, 25(4), 369-375. HERO ID: 58035

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score



9. Reporting of

Doses/
Concentrations

Actual concentrations
were not reported.
Concentrations 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
concentrations. Due to
the lack of reporting of
actual concentrations for

vapor exposures, 1
downgraded this metric
to low.

Low

3

2

6

10. Exposure
Frequency and
Duration

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

High

1

1

1

11. Number of
Exposure Groups
and Dose Spacing

The number of exposure
groups and
dose/concentration
spacing were adequate
to address the purpose
of the study. Selected
concentrations were not

justified by the study
authors but the range of
concentrations was
appropriate.

High

1

1

1

24

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

12. Exposure Route
and Method

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 1
downgraded the score to
low.

Low

Test Organism

13. Test Animal
Characteristics

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

Medium

14. Adequacy and

Consistency of
Animal Husbandry
Conditions

Husbandry conditions
(temperature, humidity,

light cycle) were not
sufficiently reported to
evaluate if husbandry
was adequate and
similar among the
groups, so 1 downgraded
the score for this metric
to low.

Low

15. Number per
Group

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
substantial impact on
results.

Medium

-------
PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Outcome
Assessment

16. Outcome
Assessment
Methodology

The outcome assessment

methodology was
reported and specific for
the outcomes of interest
(neurobehavioral
effects). However, the
study did not include a

post-mortem
examination of neural
tissue.

Medium

17. Consistency of
Outcome
Assessment

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
effect; however, not all
time points evaluated
were reported.

Low

18. Sampling
Adequacy

Details regarding
sampling were not
reported to determine if
sampling was adequate

for all groups. For
example, it's not stated
how many of the eight
animals per group were
evaluated, neither in the
text nor in the results
table (Table IV).

Low

-------
PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

19. Blinding of
Assessors

Blinding status was not
reported in this study.

Neurobehavioral
assessments typically
need to be conducted 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.

Low

20. Negative Control
Response

Negative control data
were not shown for all
outcomes; however,
negative control data

were compared to
treatment groups for
purposes of determining
effects on evaluated
outcomes (e.g., body
weight, avoidance
response, escape
response, as shown in
Table IV). These
uncertainties are unlikely
to have a substantial
impact on results.

Low

Confounding /
Variable Control

21. Confounding
Variables in Test
Design and
Procedures

There were no
confounding differences

reported among the
study groups; however,
initial body weight or
food/water intake were

not reported.
Additionally, respiratory
rate was not reported,
but 1,4-dioxane is a
potential respiratory
irritant, so 1 scored this
metric as low.

Low

-------
PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

22. Health
Outcomes Unrelated
to Exposure

Data on attrition and

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

Medium

Data Presentation
and Analysis

23. Statistical
Methods

Statistical methods were
reported for body weight

data, but not for
evaluation of avoidance

and escape response
data. Mean values with
standard deviations
were not reported for
avoidance and escape
response data, so an
independent analysis
would not be possible.

Low

24. Reporting of
Data

Body weight effects
were reported (e.g.,
Table IV) but data were

not shown in full.
Neurological/behavioral
effects, as reported in
Table IV, were observed,
but data were not
reported completely
(only %'s affected are
shown).

Low

High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3

Sum of scores:

Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:

2.2333

Overall Score:
Nearest *:

2.2

Overall Quality Level:

Medium

-------
PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

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

Study reference:

Mattie, D. R.,Bucher, T. W.,Carter, A. L.,Stoffregen, D. E.,Reboulet, J. E. (2012). Acute Inhalation Toxicity Study of
1, 4-Dioxane in Rats (Rattus norvegicus) GRAand 1(20), 29. HERO ID: 3563367

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Test Substance

1. Test Substance
Identity

Clearly identified: 1,4-

dioxane ((formula:
C4H802); CAS # 123-91-

High

2. Test Substance
Source

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

Medium

3. Test Substance
Purity

>99% purity

High

Test Design

4. Negative and
Vehicle Controls

Concurrent negative
controls were exposed
to clean air.

High

5. Positive Controls

Positive control not
required for study type
(OECD 412)

Not Rated

6. Randomized
Allocation

Animals were "randomly
selected for each
exposure group".

High

Exposure
Characterization

7. Preparation and
Storage of Test
Substance

Vapor generation
method was adequately
reported.

High

8. Consistency of

Exposure
Administration

Exposure methods were
consistent between
groups.

High

9. Reporting of

Doses/
Concentrations

Target and analytical
concentrations reported

(Table 4). Exposure
chamber concentrations
were continuously
sampled and the
concentration
determined
approximately every 40
seconds by FTIR analysis
for each entire 6 hour
exposure.

High

10. Exposure
Frequency and
Duration

Exposure duration
consistent with cited
guideline (OECD 412)

High

-------
PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Mattie, D. R.,Bucher, T. W.,Carter, A. L.,Stoffregen, D. E.,Reboulet, J. E. (2012). Acute Inhalation Toxicity Study of
1, 4-Dioxane in Rats (Rattus norvegicus) GRA and 1(20), 29. HERO ID: 3563367

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score



11. Number of
Exposure Groups
and Dose Spacing

Three exposure groups
plus concurrent controls
were used (consistent
with guideline (OECD
412).. Methods section
states that exposure
levels were based on
levels in the
accompanying acute (6-
hr) study). However, the
discussion states that
based on a general 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.

High

1

1

1

12. Exposure Route
and Method

Dynamic, whole-body
exposure with 15
complete fresh air
changes per hour;
individually housed in
690 L chambers. Any
aerosols that were

formed during
vaporization process
were captured by a
patch of glass wool
upstream, so nose-only
exposure was not
necessary.

High

1

1

1

Test Organism

13. Test Animal
Characteristics

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.

High

1

2

2

30

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Mattie, D. R.,Bucher, T. W.,Carter, A. L.,Stoffregen, D. E.,Reboulet, J. E. (2012). Acute Inhalation Toxicity Study of
1, 4-Dioxane in Rats (Rattus norvegicus) GRA and 1(20), 29. HERO ID: 3563367

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

14. Adequacy and

Consistency of
Animal Husbandry
Conditions

Husbandry conditions
were the same between

groups. All animals
acclimated to exposure
chambers for 5 days
before exposure.

High

15. Number per
Group

16/sex/group; 8/sex
sacrificed at end of
exposure, 8/sex
sacrificed 2 weeks after

exposure (minimum
guideline: 5/sex/group
per sacrifice)

High

16. Outcome
Assessment
Methodology

Body weight- at
randomization, before
each exposure, weekly
during recovery, at
necropsy
Clinical signs of
neurotoxicity (autonomic
effects, central nervous

system effects, and
reactivity to handling or
sensory stimuli)

High

Outcome

17. Consistency of
Outcome
Assessment

Assessment identical
across groups.

High

Assessment

18. Sampling
Adequacy

Sampling consisted with
cited guideline (OECD
412)

High

19. Blinding of
Assessors

No reporting of blinding
status of examiners
during subjective
assessments of clinical
signs of neurotoxicity.

Unacceptable

20. Negative Control
Response

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

Unacceptable

-------
PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Mattie, D. R.,Bucher, T. W.,Carter, A. L.,Stoffregen, D. E.,Reboulet, J. E. (2012). Acute Inhalation Toxicity Study of
1, 4-Dioxane in Rats (Rattus norvegicus) GRA and 1(20), 29. HERO ID: 3563367

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score



21. Confounding
Variables in Test
Design and
Procedures

Methods section states
that evaluations of
respiration 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
respiratory irritant.

Low

3

2

6

Confounding /
Variable Control

22. Health
Outcomes Unrelated
to Exposure

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), 1 rated
as medium.

Medium

2

1

2

Data Presentation
and Analysis

23. Statistical
Methods

No mention of statistical

analysis of clinical
neurotoxicity evaluation
(data not reported).
Body weight was
reportedly analyzed with
Student's t-test and
ANOVA (data not
reported)

Unacceptable

4

1

4



24. Reporting of
Data

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

Unacceptable

4

2

8





Sum of scores:



30

51

High: >=1 and <1.7
Medium: >=1.7 and <2.3

Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:

1.7

Overall Score
(Rounded):

1.71

Low: >=2.3 and <=3















Overall Quality Level:



Unacceptable1



32

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Mattie, D. R.,Bucher, T. W.,Carter, A. L.,Stoffregen, D. E.,Reboulet, J. E. (2012). Acute Inhalation Toxicity Study of
1, 4-Dioxane in Rats (Rattus norvegicus) GRA and 1(20), 29. HERO ID: 3563367

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Footnote:

1 Consistent with our Application of Systematic Review in TSCA Risk 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, four of the metrics were rated as
unacceptable. As such, the study is considered unacceptable and the score is presented solely to increase transparency.

33

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

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

Study reference:

Mattie, D. R.,Bucher, T. W.,Carter, A. L.,Stoffregen, D. E.,Reboulet, J. E. (2012). Acute Inhalation Toxicity Study of
1, 4-Dioxane in Rats (Rattus norvegicus) GRAand 1(20), 29. HERO ID: 3563367

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Test Substance

1. Test Substance
Identity

Clearly identified: 1,4-

dioxane ((formula:
C4H802); CAS # 123-91-

High

2. Test Substance
Source

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

Medium

3. Test Substance
Purity

>99% purity

High

Test Design

4. Negative and
Vehicle Controls

Concurrent negative
controls were exposed
to clean air.

High

5. Positive Controls

Positive control not
required for study type
(OECD 412)

Not Rated

6. Randomized
Allocation

Animals were "randomly
selected for each
exposure group".

High

Exposure
Characterization

7. Preparation and
Storage of Test
Substance

Vapor generation
method was adequately
reported.

High

8. Consistency of

Exposure
Administration

Exposure methods were
consistent between
groups.

High

9. Reporting of

Doses/
Concentrations

Target and analytical
concentrations reported

(Table 4). Exposure
chamber concentrations
were continuously
sampled and the
concentration
determined
approximately every 40
seconds by FTIR analysis
for each entire 6 hour
exposure.

High

10. Exposure
Frequency and
Duration

Exposure duration
consistent with cited
guideline (OECD 412)

High

-------
PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Mattie, D. R.,Bucher, T. W.,Carter, A. L.,Stoffregen, D. E.,Reboulet, J. E. (2012). Acute Inhalation Toxicity Study of
1, 4-Dioxane in Rats (Rattus norvegicus) GRA and 1(20), 29. HERO ID: 3563367

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score



11. Number of
Exposure Groups
and Dose Spacing

Three exposure groups
plus concurrent controls
were used (consistent
with guideline (OECD
412).. Methods section
states that exposure
levels were based on
levels in the
accompanying acute (6-
hr) study). However, the
discussion states that
based on a general 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.

High

1

1

1

12. Exposure Route
and Method

Dynamic, whole-body
exposure with 15
complete fresh air
changes per hour;
individually housed in
690 L chambers. Any
aerosols that were

formed during
vaporization process
were captured by a
patch of glass wool
upstream, so nose-only
exposure was not
necessary.

High

1

1

1

Test Organism

13. Test Animal
Characteristics

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.

High

1

2

2

35

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Mattie, D. R.,Bucher, T. W.,Carter, A. L.,Stoffregen, D. E.,Reboulet, J. E. (2012). Acute Inhalation Toxicity Study of
1, 4-Dioxane in Rats (Rattus norvegicus) GRA and 1(20), 29. HERO ID: 3563367

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

14. Adequacy and

Consistency of
Animal Husbandry
Conditions

Husbandry conditions
were the same between

groups. All animals
acclimated to exposure
chambers for 5 days
before exposure.

High

15. Number per
Group

16/sex/group; 8/sex
sacrificed at end of
exposure, 8/sex
sacrificed 2 weeks after

exposure (minimum
guideline: 5/sex/group
per sacrifice)

High

Outcome
Assessment

16. Outcome
Assessment
Methodology

Hepatic, Renal - Clinical

chemistry, OW, HP
Respiratory - HP of entire

respiratory tract,
including 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

High

17. Consistency of
Outcome
Assessment

Assessment identical
across groups.

High

18. Sampling
Adequacy

Sampling consisted with
cited guideline (OECD
412)

High

19. Blinding of
Assessors

Only non-subjective
outcomes and initial
histopathological
evaluations performed;
blinding not necessary.

Not Rated

-------
PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Mattie, D. R.,Bucher, T. W.,Carter, A. L.,Stoffregen, D. E.,Reboulet, J. E. (2012). Acute Inhalation Toxicity Study of
1, 4-Dioxane in Rats (Rattus norvegicus) GRA and 1(20), 29. HERO ID: 3563367

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

20. Negative Control
Response

Quantitative lesion data

reported. Qualitative
statement regarding no
statistically significant
changes in clinical
chemistry or hematology
covers both control and
exposure groups. Organ
weight data not reported
for any group
(downgraded in data
presentation metric, not
here)

High

Confounding /
Variable Control

21. Confounding
Variables in Test
Design and
Procedures

Low

22. Health
Outcomes Unrelated
to Exposure

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), 1 rated
as medium.

Medium

Data Presentation
and Analysis

23. Statistical
Methods

Lesion incidence
compared with Fisher's
exact test. Continuous
data analyzed by t-test
and ANOVA.

High

-------
PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Mattie, D. R.,Bucher, T. W.,Carter, A. L.,Stoffregen, D. E.,Reboulet, J. E. (2012). Acute Inhalation Toxicity Study of
1, 4-Dioxane in Rats (Rattus norvegicus) GRA and 1(20), 29. HERO ID: 3563367

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score



24. Reporting of
Data

Quantitative reporting of

lesions. Qualitative
negative result reporting

for hematology and
clinical chemistry. Organ
weights not reported.

Likely no effect (no
impact on outcome), so
rated as medium.

Medium

2

2

4

High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3

Sum of scores:



29

37

Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:

1.2759

Overall Score:
Nearest *:

1.3

Overall Quality Level:

High

38

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

3. Subchronic and Chronic Toxicity Studies (Including Cancer)

3.1. Animal toxicity evaluation results of Kano et al 2008 for a 13-week oral toxicity of 1,4-d in rats and

mice study

Study reference:

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.
HERO ID: 196245

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Test Substance

1. Test Substance
Identity

Test substance identified
by name; no concern
with different forms or
mixtures.

High

1

2

2

2. Test Substance
Source

Test substance obtained
from commercial source,
and its purity established
by IS and GC.

High

1

1

1

3. Test Substance
Purity

Test substance obtained
from commercial source;
purity >99.0% verified by
ISandGC.

High

1

1

1

Test Design

4. Negative and
Vehicle Controls

Control group received

vehicle (deionized
water); all groups were
body-weight matched
(stratified
randomization).

High

1

2

2

5. Positive Controls

Not indicated for study
type.

Not Rated

NA

NA

NA

6. Randomized
Allocation

Group assignments by
stratified randomization
into body-weight
matched groups.

High

1

1

1

39

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

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.
HERO ID: 196245

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Exposure
Characterization

7. Preparation and
Storage of Test
Substance

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
preparation showed
concentrations 94.6-
102.9% of target;
analysis of test material
4 days after preparation
showed concentrations
92.8-101.1% of initial
concentrations.

High

8. Consistency of

Exposure
Administration

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

High

9. Reporting of

Doses/
Concentrations

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

High

10. Exposure
Frequency and
Duration

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

High

-------
PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

11. Number of
Exposure Groups
and Dose Spacing

The rationale for dose
selection was not stated,
but the study included 5

non-zero exposure
concentrations across a
39-fold range. Exposure
levels included those
high enough to induce
effects and low enough
to identify a NOAEL.

High

12. Exposure Route
and Method

Exposure route was

reported and
appropriate (drinking
water).

High

13. Test Animal
Characteristics

Test animal species,
strain, age, and source
were all reported and
appropriate for
subchronic toxicity
evaluation.

High

Test Organism

14. Adequacy and

Consistency of
Animal Husbandry
Conditions

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

High

15. Number per
Group

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

High

Outcome
Assessment

16. Outcome
Assessment
Methodology

Outcome assessment
was described in detail
including
organs/endpoints,
methods,
instrumentation, stains,
and timing. Endpoints
evaluated were sensitive
for systemic toxicity.

High

17. Consistency of
Outcome
Assessment

No inconsistencies in
protocol execution were
noted in the report.

High

-------
PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

18. Sampling
Adequacy

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

High

19. Blinding of
Assessors

There were no subjective
outcomes evaluated.

Not Rated

20. Negative Control
Response

Adequately reported.

High

Confounding /
Variable Control

21. Confounding
Variables in Test
Design and
Procedures

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.

Unacceptable

22. Health
Outcomes Unrelated
to Exposure

Animal attrition was
limited to two deaths
(one rat and one mouse).
No infections or other

health outcomes
unrelated to exposure
were reported.

High

Data Presentation
and Analysis

23. Statistical
Methods

Statistical methods were

clearly described and
appropriate for the data.

High

24. Reporting of
Data

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

High

High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3

Sum of scores:

Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:

Overall Score:
Nearest *:

Overall Quality Level:

Medium

-------
PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

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.
HERO ID: 196245

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Study Quality
Comment:

The reviewer upgraded this study's overall quality rating, changing its status from unacceptable to acceptable.
They noted: Although there was a dose-related decrease in water intake that exceeded 20% at the highest 2-3
exposure levels, data from the lower exposure groups may be useful. All other metrics were rated high. The study
was initially assigned a rating of unacceptable (score = 4) with a calculated score of 1.2 (shown solely for
transparency). No calculated score is identified for the current rating in the table above because the study was

upgraded to medium.

43

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

3.2. 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 reference:

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. HERO ID: 195044

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Test Substance

1. Test Substance
Identity

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

High

2. Test Substance
Source

Obtained from Wako

Pure Chemical
Industries, Ltd. (Osaka,
Japan). Batch number
not provided, but
identity and composition
verified by laboratory
using GC-MS.

High

3. Test Substance
Purity

Reagent grade 1,4-
Dioxane (>99% pure);
analyzed for purity and
stability using GC-MS
before and after 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.

High

Test Design

4. Negative and
Vehicle Controls

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

High

5. Positive Controls

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

Not Rated

6. Randomized
Allocation

stratified randomization

into 8 body-weight-
matched groups, each
comprised of 10 rats/sex

High

Exposure
Characterization

7. Preparation and
Storage of Test
Substance

Detailed description of

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

High

-------
PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

8. Consistency of

Exposure
Administration

Exposure conditions
identical between
groups (except exposure
levels). All animals in an

exposure group were
exposed simultaneously
(exposure chamber held
20 individual cages).

High

9. Reporting of

Doses/
Concentrations

Analytical concentrations
reported, and within 1%

of target. Chamber
concentrations of 1,4-
dioxane monitored every

15 minutes during
exposure. Accuracy 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.

High

10. Exposure
Frequency and
Duration

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

High

11. Number of
Exposure Groups
and Dose Spacing

Adequate number of
exposure groups (n=7
plus control). However,

lowest dose was
identified as a LOAEL (no
NOAEL identified), and
the highest dose was
100% lethal (high dose
too high). However, the
number of dose groups
provides dose response

data (increased
effects/incidence with
increasing dose).

Medium

-------
PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

12. Exposure Route
and Method

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

High

Test Organism

13. Test Animal
Characteristics

Six-week-old F344/DuCrj

rats of both sexes
(obtained at 4-weeks of
age)

High

14. Adequacy and

Consistency of
Animal Husbandry
Conditions

Housing conditions
described adequately;

same conditions in
control and exposure
groups.

High

15. Number per
Group

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

High

Outcome
Assessment

16. Outcome
Assessment
Methodology

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

High

-------
PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

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. HERO ID: 195044

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score



17. Consistency of
Outcome
Assessment

Outcomes were assessed
consistently across study
groups as described in
methods section with
exception 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
exposure 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.

High

1

1

1

18. Sampling
Adequacy

Sampling consistent with
cited OECD guideline
413.

High

1

1

1

19. Blinding of
Assessors

Blinding status of
assessors was not
reported, Evaluated
endpoints included non-
subjective metrics and
initial histopathology
review, so blinding was
not needed.

Not Rated

NA

NA

NA

20. Negative Control
Response

Control results were
reported, and within
expected biological
variation.

High

1

1

1

47

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

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. HERO ID: 195044

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Confounding /
Variable Control

21. Confounding
Variables in Test
Design and
Procedures

Initial groups were
weight-matched. No
abnormal clinical signs
were reported in
surviving groups (all
high-dose animals died

within a week), so
altered breathing with
exposure is unlikely.
However, respiratory

rate (or lack of
bradypnea) was not
specifically mentioned so
1 downgraded to
medium.

Medium

2

2

4

22. Health
Outcomes Unrelated
to Exposure

Mortality was limited to
the high-exposure group,

and was attributed to
exposure-related effects
(renal failure)

High

1

1

1

Data Presentation
and Analysis

23. Statistical
Methods

Continuous variables
were evaluated using

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

High

1

1

1

48

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

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. HERO ID: 195044

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score



24. Reporting of
Data

Only some of the blood

parameters (clinical
chemistry, 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 decrease in urinary
protein was qualitatively
reported; no other
urinalysis results were

reported (again,
assumed 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 histology
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
results).

Medium

2

2

4

High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3

Sum of scores:



29

34

Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:

1.1724

Overall Score:
Nearest *:

1.2

Overall Quality Level:

High

49

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

3.3. 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

Study reference:

Kociba, R. J.,McCollister, S. B.,Park, C.,Torkelson, T. R.,Gehring, P. J. (1974). 1,4-dioxane. 1. Results of a 2-year
ingestion study in rats Toxicology and Applied Pharmacology, 30(2), 275-286. HERO ID: 62929

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Test Substance

1. Test Substance
Identity

Clearly identifies
substance as 1,4-dioxane

High

2. Test Substance
Source

Compound obtained
from The Dow Chemical
Co. (batch no. not
reported).

Medium

3. Test Substance
Purity

Purity not reported, but

stock samples were
analyzed for impurities

at 6 different times
during 2-year study. The

following impurities
were reported 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%.

High

Test Design

4. Negative and
Vehicle Controls

Untreated controls were
given regular drinking
water.

High

5. Positive Controls

Positive control not
warranted by study type.

Not Rated

6. Randomized
Allocation

The study did not report

how animals were
allocated to study groups

Low

Exposure
Characterization

7. Preparation and
Storage of Test
Substance

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.

High

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

8. Consistency of

Exposure
Administration

Drinking water was
available ad libitum to all
exposure groups.

High

9. Reporting of

Doses/
Concentrations

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.

High

10. Exposure
Frequency and
Duration

2 yr study; drinking
water available ad
libitum

High

11. Number of
Exposure Groups
and Dose Spacing

3 dose groups - low dose

did not induce toxic
effects or tumors; mid-
dose induced some toxic
effects, high-dose
induced tumors.

High

12. Exposure Route
and Method

drinking water
administration

High

13. Test Animal
Characteristics

6-8 wk old Sherman rats;
male and female

High

Test Organism

14. Adequacy and

Consistency of
Animal Husbandry
Conditions

Information on
husbandry limited to
"maintained in animal
care facilities fully
accredited by the
American Association for

Accreditation of
laboratory Animal Care".
All rats were maintained
under these "approved
conditions". Water and
standard feed available
ad libitum.

Medium

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

15. Number per
Group

60/sex/group

High

Outcome
Assessment

16. Outcome
Assessment
Methodology

Cancer: complete
histological analysis,
sufficient duration of
study
Renal: OW,
histopathology
Hepatic: OW,
histopathology
Hematology, Bd wt,
mortality - adequately
assessed

High

17. Consistency of
Outcome
Assessment

The same protocols were
used for control and
exposure groups.

High

18. Sampling
Adequacy

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

High

19. Blinding of
Assessors

All evaluations were
non-subjective or initial
histopathological
evaluations.

Not Rated

20. Negative Control
Response

Control results reported,
no noted deviations
from expectation.

High

Confounding /
Variable Control

21. Confounding
Variables in Test
Design and
Procedures

Based on graphically
reported data, BW were
similar between groups
at study initiation.
Decreased water
consumption was
observed in high-dose
group (10-12% during
Days 1-198) and mid-
dose group females (8%
from days 114-198).

High

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

22. Health
Outcomes Unrelated
to Exposure

Decreased survival
during the first 4 months
of the study in the high-
dose group attributed to
exposure (hepatic and
renal toxicity); mortality

was comparable to
control in low- and mid-
dose group.

High

Data Presentation
and Analysis

23. Statistical
Methods

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.

High

24. Reporting of
Data

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),
nonneoplastic changes
reported qualitatively
only

Hematological - no
change in parameters

(qualitative)

Bd wt and Mortality
reported graphically

Medium

High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3

Sum of scores:

Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:

1.2069

Overall Score:
Nearest *:

1.2

Overall Quality Level:

High

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

3.4. 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

Study reference:

Torkelson, T. R.,Leong, B. K. J.,Kociba, R. J.,Richter, W. A.,Gehring, P. J. (1974). 1,4-Dioxane. II. Results of a 2-year
inhalation study in rats Toxicology and Applied Pharmacology, 30(2), 287-298. HERO ID: 94807

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Test Substance

1. Test Substance
Identity

The test substance was
clearly identified by
name.

High

2. Test Substance
Source

The source of the test
substance was reported.

Details regarding
analytical verification of
test substance identity
were not provided, but
are not likely to impact
the study results.

Medium

3. Test Substance
Purity

The test substance purity
was reportedly 99.9%;
therefore, any effects
observed are likely due
to the nominal test
substance.

High

Test Design

4. Negative and
Vehicle Controls

The study authors
reported using an
appropriate concurrent
negative control group
(rats exposed to filtered
air only).

High

5. Positive Controls

Positive controls not
indicated by study type.

Not Rated

6. Randomized
Allocation

The study authors did
not indicate how animals
were allocated to study
groups,

Low

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Exposure
Characterization

7. Preparation and
Storage of Test
Substance

Samples of the test
substance were padded
with nitrogen 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.

Medium

8. Consistency of

Exposure
Administration

Details of exposure
administration were
generally reported (same
exposure frequency,
consistent chamber
design). There were 4
animals per cage during

and in between
exposures; time of day of
exposures occurred was
not specified.

Medium

9. Reporting of

Doses/
Concentrations

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 analyses).

High

10. Exposure
Frequency and
Duration

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

High

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

11. Number of
Exposure Groups
and Dose Spacing

The dose groups and
spacing are not relevant
for assessment. 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
control. This study used
one group exposed to
the test substance and a
control group. The
number of exposure
groups is not adequate
to evaluate exposure-
response relationships.
The concentration of the
test substance used in
the study was based on
the threshold limit value
(ACGIH), but was not
high enough to elicit
toxicity.

Unacceptable

12. Exposure Route
and Method

Rats were exposed to
the test substance under
dynamic exposure
conditions.

High

Test Organism

13. Test Animal
Characteristics

General information
regarding test animal
characteristics (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.

Medium

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

14. Adequacy and

Consistency of
Animal Husbandry
Conditions

Husbandry conditions
were not reported in
sufficient detail to
determine if conditions

were the
same/adequate between
control and exposed
groups.

Low

15. Number per
Group

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.

High

Outcome
Assessment

16. Outcome
Assessment
Methodology

The outcome
methodology addressed
the intended outcomes
of interest.

High

17. Consistency of
Outcome
Assessment

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

High

18. Sampling
Adequacy

Endpoints (including
hematology and clinical

chemistry, gross and
microscopic pathology)
were evaluated in all
surviving animals.

High

19. Blinding of
Assessors

Blinding not required for
initial histopathology
examinations (other
endpoints evaluated
were not subjective).

Not Rated

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

20. Negative Control
Response

In general, the incidence
of tumors in control and
exposed rats was low or
none. Both treated rats

and controls showed
reticulum cell sarcomas
and mammary tumors.
The study authors

indicated that
"numerous tumors
characteristic of this
strain were seen in all
groups."

High

Confounding /
Variable Control

21. Confounding
Variables in Test
Design and
Procedures

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.

Medium

22. Health
Outcomes Unrelated
to Exposure

Data on attrition and/or

health outcomes not
related to exposure were
not reported because
there were not any
significant differences
among groups.

High

23. Statistical
Methods

Statistical methods were

described (in minimal
detail) and appear to be
appropriate.

High

Data Presentation
and Analysis

24. Reporting of
Data

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).

Medium

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Torkelson, T. R.,Leong, B. K. J.,Kociba, R. J.,Richter, W. A.,Gehring, P. J. (1974). 1,4-Dioxane. II. Results of a 2-year
inhalation study in rats Toxicology and Applied Pharmacology, 30(2), 287-298. HERO ID: 94807

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3

Sum of scores:



29

45

Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:

1.5517

Overall Score
(Rounded):

1.61

Overall Quality Level:

Unacceptable1

Footnote:

1 Consistent with our Application of Systematic Review in TSCA Risk 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 of the metrics was rated as
unacceptable. As such, the study is considered unacceptable and the score is presented solely to increase transparency.

59

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

Study reference:

Argus, M. F., Arcos, J. C.,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. HERO ID: 17009

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium
,Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Test Substance

1. Test Substance
Identity

Test substance identified
by name and chemical
formula and structure

High

2. Test Substance
Source

Eastman organic
chemical number was
reported

Medium

3. Test Substance
Purity

Purity was not reported

Low

Test Design

4. Negative and
Vehicle Controls

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
challenging.

Low

5. Positive Controls

The metric is not
applicable.

Not Rated

6. Randomized
Allocation

How animals were
allocated was not
reported.

Low

Exposure
Characterization

7. Preparation and
Storage of Test
Substance

Limited preparation (1%
in drinking water)
information 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.

Medium

8. Consistency of

Exposure
Administration

Treated animals had
access to drinking water
continuously

High

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium
,Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

9. Reporting of

Doses/
Concentrations

The maximum dose/rat,
approximate daily water

intake rate, and body
weight range at the end
of the study were
reported, so
approximation of dose
could be calculated.

Medium

10. Exposure
Frequency and
Duration

Data found in Table 1.

High

11. Number of
Exposure Groups
and Dose Spacing

Only one treatment dose
was used

Not Rated

12. Exposure Route
and Method

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

High

Test Organism

13. Test Animal
Characteristics

Animal source, species,
strain, sex, life-stage,
and body weight range
were reported. Specific
age and health status
was not reported.

Medium

14. Adequacy and

Consistency of
Animal Husbandry
Conditions

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

Medium

15. Number per
Group

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.

Medium

Outcome
Assessment

16. Outcome
Assessment
Methodology

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

Medium

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium
,Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

17. Consistency of
Outcome
Assessment

Based on the study
report, it is inferred that
outcome assessment
was consistent.

High

18. Sampling
Adequacy

Sampling was adequate.

High

19. Blinding of
Assessors

This metric is not
applicable.

Not Rated

20. Negative Control
Response

Biological responses
were adequate.

High

Confounding /
Variable Control

21. Confounding
Variables in Test
Design and
Procedures

The lack of reported of
initial body weight and
specific water intake is

not likely to have a
substantial impact on
results.

Medium

22. Health
Outcomes Unrelated
to Exposure

Data on attrition and/or

health outcomes
unrelated to exposure
were not reported.

Low

Data Presentation
and Analysis

23. Statistical
Methods

Statistical analysis was
not conducted, but some

data were provided
which could be used to
do an independent
analysis (incidence of
rats with tumors)

Low

24. Reporting of
Data

Tabular data for tumor
outcomes was reported,

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

Medium

High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3

Sum of scores:

Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:

1.9286

Overall Score:
Nearest *:

1.9

Overall Quality Level:

Medium

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

3.6. Animal toxicity evaluation results of Argus et al 1973 for a carcinogenicity-liver (dose response),

electron microscopy study on cancer outcomes

Study reference:

Argus, M. F.,Sohal, R. S.,Bryant, G. M.,Hoch-Ligeti, C.,Arcos, J. C. (1973). Dose-response and ultrastructural
alterations in dioxane carcinogenesis. Influence of methylcholanthrene on acute toxicity European Journal of
Cancer, 9(4), 237-243. HERO ID: 62912

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

1. Test Substance
Identity

Identified by name and
source same as Argus et
al., 1965 , which limits
uncertainties

Medium

Test Substance

2. Test Substance
Source

Source reported but no
additional details were
reported

Medium

3. Test Substance
Purity

Purity was not reported

Low

4. Negative and
Vehicle Controls

There were no apparent
differences in the
concurrent control
group.

High

Test Design

5. Positive Controls

This metric was not
applicable.

Not Rated

6. Randomized
Allocation

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

Low

7. Preparation and
Storage of Test
Substance

Solutions were prepared
fresh daily in drinking
water.

High

8. Consistency of

Exposure
Administration

There were no apparent

inconsistencies in
exposure administration.

High

Exposure
Characterization

9. Reporting of

Doses/
Concentrations

The doses were reported
along with average fluid
consumption

High

10. Exposure
Frequency and
Duration

Duration was provided

High

11. Number of
Exposure Groups
and Dose Spacing

The number of exposure
groups and dose spacing
were appropriate

High

12. Exposure Route
and Method

The route and method
were appropriate.

High

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Test Organism

13. Test Animal
Characteristics

The species, strain, sex,
age, initial body weight
range, and source were
reported. The health
status of the animals was
not reported.

Medium

14. Adequacy and

Consistency of
Animal Husbandry
Conditions

Husbandry conditions
were not sufficiently
reported to evaluate if
adequate.

Low

15. Number per
Group

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 specified.

Medium

Outcome
Assessment

16. Outcome
Assessment
Methodology

Limited details in
outcome assessment
methodology was
provided.

Medium

17. Consistency of
Outcome
Assessment

It is inferred that
outcome assessment
was consistent.

High

18. Sampling
Adequacy

All animals were
assessed.

High

19. Blinding of
Assessors

This metric is not
applicable.

Not Rated

20. Negative Control
Response

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

Unacceptable

Confounding /
Variable Control

21. Confounding
Variables in Test
Design and
Procedures

No differences were
reported.

High

22. Health
Outcomes Unrelated
to Exposure

Details were not
reported

Low

Data Presentation
and Analysis

23. Statistical
Methods

Statistical methods were
not reported

Low

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Argus, M. F.,Sohal, R. S.,Bryant, G. M.,Hoch-Ligeti, C.,Arcos, J. C. (1973). Dose-response and ultrastructural
alterations in dioxane carcinogenesis. Influence of methylcholanthrene on acute toxicity European Journal of
Cancer, 9(4), 237-243. HERO ID: 62912

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score



24. Reporting of
Data

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

Low

3

2

6

High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3

Sum of scores:



29

54

Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:

NA

Overall Score:
Nearest *:

NA

Overall Quality Level:

Low

Study Quality
Comment:

The reviewer upgraded this study's overall quality rating, changing its status from unacceptable to
acceptable. They noted: The study would be upgraded because a description of the tumors observed was
provided which is informative. Also, effective tumor doses were provided. The study was initially
assigned a rating of unacceptable (score = 4) with a calculated score of 1.9 (shown solely for
transparency). No calculated score is identified for the current rating in the table above because the

study was upgraded to low.

65

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3.7. 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 reference:

IBRC (1998). Two-year studies of 1,4-dioxane in F344 rats and BDF1 mice (drinking water). HERO ID: 196240

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Test Substance

1. Test Substance
Identity

Identified by name,
structure, and CASRN

High

1

2

2

2. Test Substance
Source

Source was reported but
no additional
information.

Medium

2

1

2

3. Test Substance
Purity

Purity such that effects
likely due to test
substance

High

1

1

1

Test Design

4. Negative and
Vehicle Controls

Appropriate negative
control group was
included

High

1

2

2

5. Positive Controls

Not applicable for this
study

Not Rated

NA

NA

NA

6. Randomized
Allocation

Allocation of animals
was not reported

Low

3

1

3

Exposure
Characterization

7. Preparation and
Storage of Test
Substance

Test substance was
administered in the
drinking water, but
additional details were
not reported..

Low

3

1

3

8. Consistency of

Exposure
Administration

Exposures were
consistent

High

1

1

1

9. Reporting of

Doses/
Concentrations

See footnote at end of
page.1

High

1

2

2

10. Exposure
Frequency and
Duration

See footnote at end of
page.1

High

1

1

1

11. Number of
Exposure Groups
and Dose Spacing

See footnote at end of
page.1

High

1

1

1

12. Exposure Route
and Method

See footnote at end of
page.1

High

1

1

1

1 Metrics that received a "High" rating met the criteria as discussed in the Applications of Systematic Review for
TSCA Risk Evaluation.

66

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

IBRC (1998). Two-year studies of 1,4-dioxane in F344 rats and BDF1 mice (drinking water). HERO ID: 196240

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Test Organism

13. Test Animal
Characteristics

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

Medium

2

2

4

14. Adequacy and

Consistency of
Animal Husbandry
Conditions

All husbandry conditions
were reported.

High

1

1

1

15. Number per
Group

See footnote at end of
page.1

High

1

1

1

Outcome
Assessment

16. Outcome
Assessment
Methodology

Outcome methodology
was appropriate and
sensitive

High

1

2

2

17. Consistency of
Outcome
Assessment

Outcomes were assessed
consistently

High

1

1

1

18. Sampling
Adequacy

Sampling was
appropriate

High

1

1

1

19. Blinding of
Assessors

Not applicable for this
study

Not Rated

NA

NA

NA

20. Negative Control
Response

See footnote at end of
page.1

High

1

1

1

Confounding /
Variable Control

21. Confounding
Variables in Test
Design and
Procedures

See footnote at end of
page.1

High

1

2

2

22. Health
Outcomes Unrelated
to Exposure

There were no
differences among
groups unrelated to
exposure

High

1

1

1

Data Presentation
and Analysis

23. Statistical
Methods

Statistical analyses were
reported and
appropriate

High

1

1

1

24. Reporting of
Data

Outcomes were
reported.

High

1

2

2

1 Metrics that received a "High" rating met the criteria as discussed in the Applications of Systematic Review for
TSCA Risk Evaluation.

67

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

IBRC (1998). Two-year studies of 1,4-dioxane in F344 rats and BDF1 mice (drinking water). HERO ID: 196240

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3

Sum of scores:



29

36

Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:

1.2414

Overall Score:
Nearest *:

1.2

Overall Quality Level:

High

68

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

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

Study reference:

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. HERO ID: 594539

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium
,Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Test Substance

1. Test Substance
Identity

Identified by CASRN and
each lot analyzed by IR
and GC.

High

2. Test Substance
Source

Obtained from
manufacturer.

High

3. Test Substance
Purity

>99% pure; confirmed by
GC

High

Test Design

4. Negative and
Vehicle Controls

Adequately reported

High

5. Positive Controls

Not indicated for study
type.

Not Rated

6. Randomized
Allocation

Stratified randomization;
matched by body weight

High

Exposure
Characterization

7. Preparation and
Storage of Test
Substance

Adequately reported;
prepared twice per week
and stable at 4 days
post-preparation.

High

8. Consistency of

Exposure
Administration

Drinking water available
to all animals ad libitum

High

9. Reporting of

Doses/
Concentrations

Data provided on water

consumption; no
difference across groups.

High

10. Exposure
Frequency and
Duration

Consistent with test
guideline for study type.

High

11. Number of
Exposure Groups
and Dose Spacing

Highest dose chosen so
as not to exceed the
MTD.

High

12. Exposure Route
and Method

Adequately reported.
Consistent with test
guideline for study type.

High

Test Organism

13. Test Animal
Characteristics

Adequately reported.
Consistent with test
guidelines for study type.

High

14. Adequacy and

Consistency of
Animal Husbandry
Conditions

Adequately reported.
Consistent with test
guidelines for study type.

High

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium
,Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

15. Number per
Group

50/sex/group; consistent
with test guidelines for
study type.

High

Outcome
Assessment

16. Outcome
Assessment
Methodology

Consistent with test
guidelines for study type.

High

17. Consistency of
Outcome
Assessment

No anomalies reported.

High

18. Sampling
Adequacy

Consistent with test
guidelines for study type.

High

19. Blinding of
Assessors

Not addressed.

Low

20. Negative Control
Response

Adequately reported; no
unusual results.

High

Confounding /
Variable Control

21. Confounding
Variables in Test
Design and
Procedures

Body-weight matching;
no difference in
food/water
consumption.

High

22. Health
Outcomes Unrelated
to Exposure

Attrition was related to
exposure.

High

Data Presentation
and Analysis

23. Statistical
Methods

Appropriate methods
chosen; adequately
reported.

High

24. Reporting of
Data

Multiple data tables
summarize all endpoints.

High

High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3

Sum of scores:

Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:

1.0667

Overall Score:
Nearest *:

1.1

Overall Quality Level:

High

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

3.9. 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 reference:

Kasai, T.,Kano, H.,Umeda, Y.,Sasaki, T.,lkawa, 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. HERO ID: 193803

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Test Substance

1. Test Substance
Identity

The test substance was
identified definitively.

High

2. Test Substance
Source

The source of the test
substance was reported,
including manufacturer,
and its identity was
verified by analytical
methods.

High

3. Test Substance
Purity

The test chemical was
reported as reagent
grade (greater than 99%
pure) and purity was also
evaluated by the
laboratory via gas
chromatography-mass
spectrometry (GC-MS). 1
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.

Medium

Test Design

4. Negative and
Vehicle Controls

The study authors
reported using an
appropriate concurrent
negative control group.

High

5. Positive Controls

Not applicable - Positive

control group is not
indicated by study type.

Not Rated

6. Randomized
Allocation

The animals were
divided by stratified
randomization into body
weight-matched groups.

High

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Exposure
Characterization

7. Preparation and
Storage of Test
Substance

The test substance was

found to be stable
throughout the 7-month
period of storage, as
determined by gas
chromatography. The
methods and equipment
used to generate the test
substance were
appropriate.

High

8. Consistency of

Exposure
Administration

Details of exposure
administration were
reported and were
consistent among the
groups. However, 1
downgraded this to
medium because the

report does not
specifically state that
exposures occurred at
the same time of day for
all animals.

Medium

9. Reporting of

Doses/
Concentrations

Actual vapor
concentrations in the
exposure chambers were

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

High

10. Exposure
Frequency and
Duration

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

High

11. Number of
Exposure Groups
and Dose Spacing

The number of exposure
groups and
concentration spacing
were justified and
adequate for the
purpose of this study.

High

-------
PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

12. Exposure Route
and Method

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).

High

Test Organism

13. Test Animal
Characteristics

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.

Medium

14. Adequacy and

Consistency of
Animal Husbandry
Conditions

All husbandry conditions
were reported and were
adequate and consistent
among the groups and
controls.

High

15. Number per
Group

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

High

Outcome
Assessment

16. Outcome
Assessment
Methodology

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

High

17. Consistency of
Outcome
Assessment

The outcome assessment
protocol was reported;
however, the
descriptions of each
outcome methodology
do not specifically state

that some outcomes
(e.g., urine, blood) were

sampled at the same
time/day for all groups.

Low

-------
PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

18. Sampling
Adequacy

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

High

19. Blinding of
Assessors

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
examination results were
not described as a re-

evaluation so 1
considered this metric
N/A.

Not Rated

20. Negative Control
Response

The negative control
response was adequate.

High

Confounding /
Variable Control

21. Confounding
Variables in Test
Design and
Procedures

There were no reported

differences in initial
weight, 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
reported and this study,
so 1 downgraded this
metric rating to Low.

Low

22. Health
Outcomes Unrelated
to Exposure

No indications of
attrition or health
outcomes unrelated to
exposure.

High

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Kasai, T.,Kano, H.,Umeda, Y.,Sasaki, T.,lkawa, 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. HERO ID: 193803

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Data Presentation
and Analysis

23. Statistical
Methods

The statistical methods
were clearly described
and appropriate for the
data set.

High

1

1

1

24. Reporting of
Data

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 1 downgraded
the score to medium.

Medium

2

2

4

High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3

Sum of scores:



29

41

Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:

1.4138

Overall Score:
Nearest *:

1.4

Overall Quality Level:

High

75

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

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

Study reference:

NCI (1978). Bioassay of 1,4-dioxane for possible carcinogenicity. HERO ID: 62935

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Test Substance

1. Test Substance
Identity

The test substance was
identified by name and
CASRN.

High

1

2

2

2. Test Substance
Source

The source of the test
substance was reported,
including lot numbers.
The test substance (one
of two lots) was analyzed
to confirm identity and
purity (using vapor phase
chromatography and
spectrometry).

High

1

1

1

3. Test Substance
Purity

The purity (one of two
lots) was 99.9%. The test
substance was tested for
specific impurities
(sodium
diethylthiocarbamate,
and peroxide); these

impurities were
generally present at
0.001% or less. However
one lot showed peroxide
levels of 0.1% after study

completion. This
deficiency is not likely to
substantially impact the
study results.

High

1

1

1

76

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

NCI (1978). Bioassay of 1,4-dioxane for possible carcinogenicity. HERO ID: 62935

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Test Design

4. Negative and
Vehicle Controls

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
report, the weights of

low-dose male rats
differed from the body
weights of control and
high-dose animals at
study week 0.

Unacceptable

4

2

8

5. Positive Controls

Positive control group
not indicated by study
type.

Not Rated

NA

NA

NA

6. Randomized
Allocation

Animals were assigned
to control or dose groups
"according 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).

Low

3

1

3

77

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

NCI (1978). Bioassay of 1,4-dioxane for possible carcinogenicity. HERO ID: 62935

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Exposure
Characterization

7. Preparation and
Storage of Test
Substance

Test substance
preparation and storage

conditions were not
reported in exhaustive
detail ("dioxane
solutions 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 provided.

Medium

2

1

2

8. Consistency of

Exposure
Administration

Dosed water or tap
water was available ad
libitum.

High

1

1

1

9. Reporting of

Doses/
Concentrations

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
sample). The study
report indicates that
"there were wide
fluctuations in intake at
different time periods
within groups."

Low

3

2

6

78

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

NCI (1978). Bioassay of 1,4-dioxane for possible carcinogenicity. HERO ID: 62935

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score



10. Exposure
Frequency and
Duration

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.

High

1

1

1

11. Number of
Exposure Groups
and Dose Spacing

Concentrations were
chosen based on the
results of 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 concurrent (i.e.. data
for only 1 quantitative
dose group and controls
in male rats were
concurrent). 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.

Low

3

1

3

12. Exposure Route
and Method

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.

Medium

2

1

2

79

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

NCI (1978). Bioassay of 1,4-dioxane for possible carcinogenicity. HERO ID: 62935

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Test Organism

13. Test Animal
Characteristics

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
carcinogenicity (although
not the same rat strain
used in previous
studies).

High

1

2

2

14. Adequacy and

Consistency of
Animal Husbandry
Conditions

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. This
is unlikely to have had a
substantial impact on
results (there were no
indications of injuries or
death due to
overcrowding).

High

1

1

1

80

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

NCI (1978). Bioassay of 1,4-dioxane for possible carcinogenicity. HERO ID: 62935

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score



15. Number per
Group

The number of animals
per study group was
lower than the typical
number used in
carcinogenicity 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
carcinogenicity).

Medium

2

1

2

Outcome
Assessment

16. Outcome
Assessment
Methodology

Animals from all dose
groups were subjected
to gross and microscopic
pathology evaluations.
The number 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.

Medium

2

2

4

17. Consistency of
Outcome
Assessment

Surviving rats were
sacrificed at 110-117
weeks. The tissues from
some animals were not
evaluated (particularly in
animals 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.

Medium

2

1

2

81

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

NCI (1978). Bioassay of 1,4-dioxane for possible carcinogenicity. HERO ID: 62935

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score



18. Sampling
Adequacy

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 impact
the study results.

Medium

2

1

2

19. Blinding of
Assessors

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

Not Rated

NA

NA

NA

20. Negative Control
Response

The biological responses
of the negative control
groups were adequate

(showing no or low
incidences of lesions).

High

1

1

1

Confounding /
Variable Control

21. Confounding
Variables in Test
Design and
Procedures

Doses administered to

low- and high-dose
groups of rats were not

reflective of the
intended doses owing (at
least in part) to
decreased palatability
(water 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

administered
dibenzodioxin, 2,7-
dichlorobenzodioxin, and

1,2,3,4,6,7,8,9-
octachlorodibenzodioxin.

Low

3

2

6

82

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

NCI (1978). Bioassay of 1,4-dioxane for possible carcinogenicity. HERO ID: 62935

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score



22. Health
Outcomes Unrelated
to Exposure

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.

High

1

1

1

Data Presentation
and Analysis

23. Statistical
Methods

Procedures used for
statistical analyses were
described 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,
carcinogenicity data for
high-dose male rats were
compared to controls
only (and not to low-
dose males). Statistical
analyses for some

endpoints (e.g.
mortality) appear to
consider all groups of
male rats, even though

dosing was not
concurrent. Incidences
of non-neoplastic lesions
were not subjected to
statistical analyses.

Medium

2

1

2



24. Reporting of
Data

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.

High

1

2

2

83

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Study reference:

NCI (1978). Bioassay of 1,4-dioxane for possible carcinogenicity. HERO ID: 62935

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3

Sum of scores:



29

55

Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:

1.8966

Overall Score
(Rounded):

1.91

Overall Quality Level:

Unacceptable1

Footnote:

1 Consistent with our Application of Systematic Review in TSCA Risk 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 of the metrics was rated as
unacceptable. As such, the study is considered unacceptable and the score is presented solely to increase transparency.

84

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3.11. 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 reference:

NCI (1978). Bioassay of 1,4-dioxane for possible carcinogenicity. HERO ID: 62935

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Test Substance

1. Test Substance
Identity

The test substance was
identified by name and
CASRN.

High

1

2

2

2. Test Substance
Source

The source of the test
substance was reported,
including lot numbers.
The test substance (one
of two lots) was analyzed
to confirm identity and
purity (using vapor phase
chromatography and
spectrometry).

High

1

1

1

3. Test Substance
Purity

The purity (one of two
lots) was 99.9%. The test
substance was tested for
specific impurities
(sodium
diethylthiocarbamate,
and peroxide); these

impurities were
generally present at
0.001% or less. However
one lot showed peroxide
levels of 0.1% after study

completion. This
deficiency is not likely to
substantially impact the
study results.

High

2

1

2

85

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

NCI (1978). Bioassay of 1,4-dioxane for possible carcinogenicity. HERO ID: 62935

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Test Design

4. Negative and
Vehicle Controls

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.

Low

3

2

6

5. Positive Controls

Positive control group
not indicated by study
type.

Not Rated

NA

NA

NA

6. Randomized
Allocation

Animals were assigned
to control or dose groups
"according 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).

Low

3

1

3

86

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Study reference:

NCI (1978). Bioassay of 1,4-dioxane for possible carcinogenicity. HERO ID: 62935

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Exposure
Characterization

7. Preparation and
Storage of Test
Substance

Test substance
preparation and storage

conditions were not
reported in exhaustive
detail ("dioxane
solutions 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 provided.

Medium

2

1

2

8. Consistency of

Exposure
Administration

Dosed water or tap
water was available ad
libitum.

High

1

1

1

9. Reporting of

Doses/
Concentrations

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
sample). The study
report indicates that
"there were wide
fluctuations in intake at
different time periods
within groups."

Low

3

2

6

87

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Study reference:

NCI (1978). Bioassay of 1,4-dioxane for possible carcinogenicity. HERO ID: 62935

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score



10. Exposure
Frequency and
Duration

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.

High

1

1

1

11. Number of
Exposure Groups
and Dose Spacing

Concentrations were
chosen based on the
results of 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 control). The
study used two dose
groups and a control.
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.

Low

3

1

3

88

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Study reference:

NCI (1978). Bioassay of 1,4-dioxane for possible carcinogenicity. HERO ID: 62935

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score



12. Exposure Route
and Method

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.

High

2

1

2

Test Organism

13. Test Animal
Characteristics

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
carcinogenicity (although
not the same rat strain
used in previous
studies).

High

1

2

2

14. Adequacy and

Consistency of
Animal Husbandry
Conditions

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
substantial impact on
results (there were no
indications of injuries or
death due to
overcrowding).

High

2

1

2

89

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Study reference:

NCI (1978). Bioassay of 1,4-dioxane for possible carcinogenicity. HERO ID: 62935

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score



15. Number per
Group

The number of animals
per study group was
lower than the typical
number used in
carcinogenicity 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
carcinogenicity).

Medium

2

1

2



16. Outcome
Assessment
Methodology

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

Medium

2

2

4

Outcome
Assessment

17. Consistency of
Outcome
Assessment

Surviving rats and mice
were sacrificed at 110-
117 and 90-93 weeks,
respectively. The tissues
from some animals were
not evaluated
(particularly in animals

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.

Medium

2

1

2

90

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

NCI (1978). Bioassay of 1,4-dioxane for possible carcinogenicity. HERO ID: 62935

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score



18. Sampling
Adequacy

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 impact
the study results.

Medium

2

1

2

19. Blinding of
Assessors

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

Not Rated

NA

NA

NA

20. Negative Control
Response

The biological responses
of the negative control
groups were adequate

(showing no or low
incidences of lesions).

High

1

1

1

Confounding /
Variable Control

21. Confounding
Variables in Test
Design and
Procedures

Doses administered to

low- and high-dose
groups of rats and mice
were not reflective of
the intended doses
owing (at least in part) to

decreased palatability
(water consumption data

were not provided).
Initial body weights were

not explicitly reported
(weights at study week 0
were shown graphically).
Rats and mice were
housed in the same
room with rats
administered
dibenzodioxin, 2,7-
dichlorobenzodioxin, and

1,2,3,4,6,7,8,9-
octachlorodibenzodioxin.

Low

3

2

6

91

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Study reference:

NCI (1978). Bioassay of 1,4-dioxane for possible carcinogenicity. HERO ID: 62935

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score



22. Health
Outcomes Unrelated
to Exposure

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.

High

2

1

2

Data Presentation
and Analysis

23. Statistical
Methods

Procedures used for
statistical analyses were
described in detail, and
appear to be relevant for

some endpoints (i.e.
cancer; the focus of this
study). Statistical
analyses for some

endpoints (e.g.
mortality) appear to
consider all groups of
rats and mice, even
when dosing was not
necessarily concurrent.

Incidences of non-
neoplastic lesions were
not subjected to
statistical analyses.

Medium

2

1

2

24. Reporting of
Data

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.

High

2

2

4

High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3

Sum of scores:



29

58

Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:

NA

Overall Score:
Nearest *:

NA

Overall Quality Level:

Low

92

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Study reference:

NCI (1978). Bioassay of 1,4-dioxane for possible carcinogenicity. HERO ID: 62935

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Study Quality
Comment:

The reviewer downgraded this study's overall quality rating. They noted: The study has some numerous
limitations. Some of these data might be usable (if dosing permutations are permitted); namely carcinogenicity
data for female rats and male and female mice. Note: The original calculated score for this study was 1.7. This
value is not presented above because the final rating was changed based on professional judgement.

93

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4. In Vivo and In Vitro Genotoxicity Studies

These studies include acute and short-term exposure duration studies. Note that one in vivo
study includes hepatic and body weight outcomes and another includes gene expression
data.

4.1. 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 reference:

Dow Chemical, Co (1989). Differentiation of the mechanisms of oncogenicity of 1,4-dioxane and 1,3-
hexachlorobutadiene in the rat. HERO ID: 4158030

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium
,Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Test Substance

1. Test Substance
Identity

1,4-dioxane

High

2. Test Substance
Source

Baker Chemical
Company; no batch
number, but purity was
analyzed by study
laboratory

Medium

3. Test Substance
Purity

>99%

High

Test Design

4. Negative and
Vehicle Controls

Concurrent vehicle
(saline) control was used

High

5. Positive Controls

No positive control; in
vivo genotoxicity study

design indicates one
should have been used
(DMN was used in the
repeat dose study only)

Unacceptable

6. Randomized
Allocation

Animals were computer

randomized into
treatment groups in all
experiments

High

Exposure
Characterization

7. Preparation and
Storage of Test
Substance

Storage details not
reported. Mixed with
saline for gavage
administration.

High

8. Consistency of

Exposure
Administration

Exposure conditions
consistent between
groups.

High

9. Reporting of

Doses/
Concentrations

Replicate 1: 0, 100, or

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

High

10. Exposure
Frequency and
Duration

Once, sacrificed after 7 d

High

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Dow Chemical, Co (1989). Differentiation of the mechanisms of oncogenicity of 1,4-dioxane and 1,3-
hexachlorobutadiene in the rat. HERO ID: 4158030

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium
,Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

11. Number of
Exposure Groups
and Dose Spacing

2-3 doses plus negative
control (two replicates)

High

12. Exposure Route
and Method

No rationale was
provided for switching
from gavage (this study)
to repeat-dose drinking

water study
(accompanying study).
Other compounds
(HCBD, DMN) were
administered via gavage
for both studies.
However, BWG was
decreased by ~45-55%
following single gavage
administration of 1000
mg/kg; this BW effect
was not observed with

drinking water
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.

Medium

Test Organism

13. Test Animal
Characteristics

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

High

14. Adequacy and

Consistency of
Animal Husbandry
Conditions

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

Medium

15. Number per
Group

4/group

High

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Dow Chemical, Co (1989). Differentiation of the mechanisms of oncogenicity of 1,4-dioxane and 1,3-
hexachlorobutadiene in the rat. HERO ID: 4158030

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium
,Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

16. Outcome
Assessment
Methodology

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

High

Outcome
Assessment

17. Consistency of
Outcome
Assessment

Consistent evaluation
across study groups

High

18. Sampling
Adequacy

4/group

High

19. Blinding of
Assessors

Only non-subjective and

initial histological
evaluations; blinding not
required.

Not Rated

20. Negative Control
Response

negative control
response was reported;

no deviations from
normal were reported.

High

Confounding /
Variable Control

21. Confounding
Variables in Test
Design and
Procedures

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
consumption (magnitude
not reported) associated
with decreased BW. This
may be the reason that
drinking water route was
used for repeat-dose

study (same high
exposure dose level).

Unacceptable

22. Health
Outcomes Unrelated
to Exposure

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.

Low

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Dow Chemical, Co (1989). Differentiation of the mechanisms of oncogenicity of 1,4-dioxane and 1,3-
hexachlorobutadiene in the rat. HERO ID: 4158030

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium
,Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Data Presentation
and Analysis

23. Statistical
Methods

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

Medium

2

1

2

24. Reporting of
Data

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

Medium

2

2

4

High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3

Sum of scores:



30

47

Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:

1.5667

Overall Score
(Rounded):

1.61

Overall Quality Level:

Unacceptable1

Footnote:

1 Consistent with our Application of Systematic Review in TSCA Risk 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, two of the metrics were rated as
unacceptable. As such, the study is considered unacceptable and the score is presented solely to increase transparency.

97

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4.2. Animal toxicity evaluation results of Itoh 2019 - in vivo genotoxicity assay - micronucleus test

Study reference:

S. Itoh (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. HERO ID:
5072318

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Test Substance

1. Test Substance
Identity

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

High

2. Test Substance
Source

Wako Pure Chemical
Industries, Ltd. (Osaka,
Japan)

High

3. Test Substance
Purity

The purity and/or grade
were not reported

Low

Test Design

4. Negative and
Vehicle Controls

Concurrent vehicle
control

High

5. Positive Controls

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

Bone marrow
micronucleus:
cyclophosphamide
monohydrate [CP]
Pig-a assay: 7,12-
dimethylbenz[a]anthrace
ne [DMBA]

High

6. Randomized
Allocation

The study did not report

how animals were
allocated to study groups

Low

Exposure
Characterization

7. Preparation and
Storage of Test
Substance

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.

High

8. Consistency of

Exposure
Administration

Exposure administration
was consistent across
exposure groups for
each experiment.

High

9. Reporting of

Doses/
Concentrations

0, 1000, 2000, or 3000
mg/kg

High

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

10. Exposure
Frequency and
Duration

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
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

High

11. Number of
Exposure Groups
and Dose Spacing

0, 1000, 2000, or 3000
mg/kg based on previous
reports

High

12. Exposure Route
and Method

Gavage at dose volume
of 10 mL/kg

High

Test Organism

13. Test Animal
Characteristics

Male F344/DuCrlCrlj rats,
4- to 8-wks of age;
Charles River
Laboratories Japan, Inc.

High

14. Adequacy and

Consistency of
Animal Husbandry
Conditions

This study was
conducted in compliance
with the following law
and

guidelines; "Law
Concerning the
Protection and Control

of Animals",
Japanese Law No. 105,
October 1, 1973, revised
on June 22, 2005

High

15. Number per
Group

4-5/group per test

High

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PEER REVIEW DRAFT, DO NOT CITE OR QUOTE

Study reference:

S. Itoh (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. HERO ID:
5072318

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Outcome
Assessment

16. Outcome
Assessment
Methodology

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)

High

1

2

2

17. Consistency of
Outcome
Assessment

See footnote at end of
page.3

High

1

1

1

18. Sampling
Adequacy

See footnote at end of
page.1

High

1

1

1

19. Blinding of
Assessors

All quantitative
measures

Not Rated

NA

NA

NA

20. Negative Control
Response

See footnote at end of
page.1

High

1

1

1

Confounding /
Variable Control

21. Confounding
Variables in Test
Design and
Procedures

Initial BW not reported;
not likely to have
substantial impact

Medium

2

2

4

22. Health
Outcomes Unrelated
to Exposure

See footnote at end of
page.1

High

1

1

1

3 Metrics that received a "High" rating met the criteria as discussed in the Applications of Systematic Review for
TSCA Risk Evaluation.

100

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Study reference:

S. Itoh (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. HERO ID:
5072318

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Data Presentation
and Analysis

23. Statistical
Methods

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

High

1

1

1

24. Reporting of
Data

Graphical reporting of all
genotox data;
quantitative reporting
for relative liver weight
data

High

1

2

2

High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3

Sum of scores:



30

36

Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:

1.2

Overall Score:
Nearest *:

1.2

Overall Quality Level:

High

101

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4.3. Animal toxicity evaluation results of Uno et al 1994 for an acute oral study on mechanistic (gene

expression/omics, genotoxicity) outcomes

Study reference:

Uno, Y.,Takasawa, H.,Miyagawa, M.,lnoue, 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. HERO ID: 194385

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

1. Test Substance
Identity

Test substance was
identified definitively.

High

Test Substance

2. Test Substance
Source

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

Medium

3. Test Substance
Purity

Purity was not reported.

Low

Test Design

4. Negative and
Vehicle Controls

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/administratio
n of vehicle, i.e., see
Table 1).

Low

5. Positive Controls

Not Rated

6. Randomized
Allocation

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

Low

Exposure
Characterization

7. Preparation and
Storage of Test
Substance

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

Low

8. Consistency of

Exposure
Administration

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

High

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Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

9. Reporting of

Doses/
Concentrations

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.

Medium

10. Exposure
Frequency and
Duration

Exposure frequency and
duration were reported
(single exposure with
evaluation at up to 48
hours post-exposure..
These appear acceptable

for the intended
outcomes for the study
(mechanistic).

High

11. Number of
Exposure Groups
and Dose Spacing

The number of exposure
groups and dose spacing

were considered
adequate to address the
purpose of the study and
were justified by the
study authors (were
based on the MTD).

High

12. Exposure Route
and Method

The exposure route and
method were reported
and were considered
appropriate for the
purpose of the study.

High

Test Organism

13. Test Animal
Characteristics

The test animal species,

strain, age, sex, and
source were reported;
however, body weight
and health status at the
start of the study were
not reported.

Medium

14. Adequacy and

Consistency of
Animal Husbandry
Conditions

Most husbandry
conditions (temperature
and light) were reported
and were similar for all
groups. Humidity was
not reported.

Medium

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Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

15. Number per
Group

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.

Medium

Outcome
Assessment

16. Outcome
Assessment
Methodology

The outcome assessment
methodology was
reported and was
sensitive for the
outcomes of interest
although it's not clear
that the duration (up to
48 hours post-exposure)
was sufficient to address
the intended outcomes.

Medium

17. Consistency of
Outcome
Assessment

The outcome assessment
methodology appeared
to be consistent among
the groups.

High

18. Sampling
Adequacy

Sampling methods
appear to have been
appropriate for
addressing the outcomes

of interest (2000
hepatocytes/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).

Medium

19. Blinding of
Assessors

No subjective outcomes
were evaluated in this
study.

Not Rated

20. Negative Control
Response

Biological responses of
the negative control
group were adequate.

High

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Study reference:

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

Confounding /
Variable Control

21. Confounding
Variables in Test
Design and
Procedures

No confounding
variables in test design
were reported; however,
initial body weight and
food/water intake were
not reported.

Medium

22. Health
Outcomes Unrelated
to Exposure

Data on attrition and

health outcomes
unrelated to exposure
for each study group
were not reported
because only differences
among groups for the
evaluated outcomes
were noted.

Medium

Data Presentation
and Analysis

23. Statistical
Methods

Statistical methods were

reported and were
appropriate for the data
sets.

High

24. Reporting of
Data

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

High

High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3

Sum of scores:

Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:

1.7931

Overall Score:
Nearest *:

1.8

Overall Quality Level:

Medium

105

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4.4 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 reference:

Dow Chemical, Co (1989). Differentiation of the mechanisms of oncogenicity of 1,4-dioxane and 1,3-
hexachlorobutadiene in the rat. HERO ID: 4158030

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

1. Test Substance
Identity

Reported only as "1,4-
dioxane".

Low

Test Substance

2. Test Substance
Source

Baker Chemical
Company; no batch
number, but purity was
analyzed by study
laboratory

Medium

3. Test Substance
Purity

>99%

High

4. Negative and
Vehicle Controls

Concurrent vehicle
(saline) control was used

High

Test Design

5. Positive Controls

Known genotoxic agent
dimethylnitrosamine
(DMN) was used as a
positive control

High

6. Randomized
Allocation

Animals were computer

randomized into
treatment groups in all
experiments

High

7. Preparation and
Storage of Test
Substance

Storage details not
reported. Mixed with

drinking water. No
details on frequency of
drinking water
preparation.

Low

8. Consistency of

Exposure
Administration

Exposure conditions
consistent between
groups.

High

Exposure
Characterization

9. Reporting of

Doses/
Concentrations

Study authors report
drinking water provided
an average 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.

Low

10. Exposure
Frequency and
Duration

11 weeks, 7d/wk

High

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Study reference:

Dow Chemical, Co (1989). Differentiation of the mechanisms of oncogenicity of 1,4-dioxane and 1,3-
hexachlorobutadiene in the rat. HERO ID: 4158030

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

11. Number of
Exposure Groups
and Dose Spacing

2 doses plus negative
and positive control

High

12. Exposure Route
and Method

No rationale was
provided for switching

from gavage
(accompanying acute
study) to repeat-dose
drinking water study.

Other compounds
(HCBD, DMN) were
administered via gavage
for both studies.
However, BWG was
decreased by ~45-55%
following single gavage
administration of 1000
mg/kg; this BW effect
was not observed with

drinking water
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.

Medium

Test Organism

13. Test Animal
Characteristics

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

High

14. Adequacy and

Consistency of
Animal Husbandry
Conditions

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

Medium

15. Number per
Group

5-6/group

High

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Study reference:

Dow Chemical, Co (1989). Differentiation of the mechanisms of oncogenicity of 1,4-dioxane and 1,3-
hexachlorobutadiene in the rat. HERO ID: 4158030

Domain

Metric

Evaluator's Comment

Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]

Metric
Score

Metric Weighting
Factor

Weighted
Score

16. Outcome
Assessment
Methodology

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

High

Outcome
Assessment

17. Consistency of
Outcome
Assessment

Consistent evaluation
across study groups

High

18. Sampling
Adequacy

5-6/group

High

19. Blinding of
Assessors

Only non-subjective and

initial histological
evaluations; blinding not
required.

Not Rated

20. Negative Control
Response

negative control
response was reported;

no deviations from
normal were reported.

High

21. Confounding
Variables in Test
Design and
Procedures

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

High

Confounding /
Variable Control

22. Health
Outcomes Unrelated
to Exposure

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

Medium

Data Presentation
and Analysis

23. Statistical
Methods

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

Medium

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