Screening-level Hazard Characterization Sponsored Chemical Ring-Substituted Anilines Category 2,6-Diethylaniline CASRN 579-66-8 2-Ethylaniline CASRN 578-54-1 Supporting Chemicals

U.S. Environmental Protection Agency
Hazard Characterization Document

December, 2012

SCREENING-LEVEL HAZARD CHARACTERIZATION
OF HIGH PRODUCTION VOLUME CHEMICALS

Ring-Substituted Anilines Category

Sponsored Chemicals

Benzenamine, 2,6-diethyl-
Benzenamine, 2-ethyl-

Structural Formula

nh2

—-CH,

H3C^J

SMILES: Nc(c(cccl)CC)clCC

nh2

SMILES: Nc(c(cccl)CC)cl

Summary

The ring substituted anilines are composed of two substances: benzenamine, 2,6-diethyl- and
benzenamine, 2-ethyl-. Benzenamine, 2,6-diethyl- is a liquid with low vapor pressure and
moderate water solubility, while benzenamine, 2-ethyl- is a liquid with low vapor pressure and
high water solubility. Both substances are expected to have moderate mobility in soil. A
supporting chemical, benzenamine, 2-ethyl-6-methyl- was not readily biodegradable in two
standard OECD screening tests and benzenamine, 2,6-diethyl- did not degrade in 14 days in
pond water, but was transformed in soil/water slurries. Volatilization is expected to be
moderate for these substances. However, they will partially exist in their protonated form
(conjugate acid) rather than as the free base at environmental pH, and the cations will not
volatilize. The rate of hydrolysis is negligible. The rate of atmospheric photooxidation is rapid.
Benzenamine, 2,6-diethyl- and benzenamine, 2-ethyl-are expected to have moderate to high
persistence (P2-P3) and low bioaccumulation potential (Bl). This assessment is based on
submitted information and limited database and literature review. A detailed review of the
published literature may reveal additional fate information.

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Acute oral toxicity of 2,6-diethylaniline and 2-ethylaniline in rats is low; acute inhalation
toxicity in rats is moderate and acute dermal toxicity in rabbits is moderate. In a 90-day dietary
repeated-dose toxicity study of 2,6-diethylaniline in rats, increased cholesterol and decreased
liver weights were noted >16.5 mg/kg-bw/day. The NOAEL is not established. In the 28-day
repeated-dose dermal toxicity study of 2,6-diethylaniline in rats, decreased body weight gain,
increased liver weights and increased serum enzyme levels (serum glutamic oxaloacetate
transaminase, (SGOT) and serum glutamic pyruvic transaminase (SGPT)) were observed at
> 300 mg/kg-day. The NOAEL is 100 mg/kg-day.

No reproductive toxicity studies are available for either chemical. In repeated-dose toxicity
studies of 2,6-diethylaniline, no effects on reproductive organs were noted. In a prenatal
developmental toxicity study of 2,6-diethylaniline in rats, maternal toxicity (salivation,
anogenital staining and decreased body weight gains) was seen at >50 mg/kg-day; the NOAEL
for maternal toxicity is not established. In this study, the developmental effects included
decreased fetal body weight and increased incidence of unossified sternebrae at 500 mg/kg-day;
the NOAEL for developmental toxicity is 250 mg/kg-day.

2,6-Diethylaniline was mutagenic showing weak response but 2-Ethylaniline was not
mutagenic in bacteria in vitro. 2,6-Diethylaniline did not induce micronuclei in mice
in vivo. 2,6-Diethylaniline was irritating to rabbit skin and irritating to rabbit eyes.

2-Ethylaniline was not irritating to rabbit skin; but was irritating to rabbit eyes.

2,6-Diethyaniline is not sensitizing to guinea pigs. 2,6-Diethylaniline did not increase tumor
incidence in rats.

Ecotoxicity of the ring-substituted anilines category members show the 96-hour LC50 for fish is
24 mg/L, the 48-h EC50 for aquatic invertebrates ranges from 8 to 21 mg/L and the 96-h EC50 for
aquatic plants is 38 mg/L for growth rate.

No data gaps were identified under the HPV Challenge Program.

The sponsor, Albemarle Corporation, submitted a Test Plan and Robust Summaries to EPA for
the ring-substituted anilines category on May 5, 2006. EPA posted the submission on the
ChemRTK HPV Challenge website on May 17, 2006

(http://www.epa.gov/oppt/chemrtk/pubs/summaries/ringsac ;.ht.m). EPA comments on

the original submission were posted to the website on December 15, 2008. Public comments
were also received and posted to the website. The sponsor submitted responses to EPA
comments and a revised test plan and robust summaries on June 29, 2009.

Category Justification

The category consists of two substances that are structurally similar with similar toxicity. EPA
considered this grouping acceptable.

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Hazard Characterization Document

December, 2012

Justification for Supporting Chemicals

The sponsor proposed aniline (CAS No. 62-53-3) and 2-ethyl-6-methylaniline

(CAS No. 24549-06-2) as supporting chemicals. EPA considered the use of these chemicals

appropriate based on their structural similarity to 2,6-diethylaniline and 2-ethylaniline.

However, in the revised submission, the sponsor submitted human health and ecotoxicity data for

their sponsored chemicals. Therefore, the supporting chemicals data are not used in this hazard

characterization.

1. Chemical Identity

1.1 Identification and Purity

The ring substituted anilines are composed of two substances: benzenamine, 2,6-diethyl- and
benzenamine, 2-ethyl-. Benzenamine, 2,6-diethyl- is a liquid with low vapor pressure and
moderate water solubility, while benzenamine, 2-ethyl- is a liquid with low vapor pressure and
high water solubility. These substances are used as chemical intermediates in the synthesis of a
variety of organic chemicals.

1.2 Physical-Chemical Properties

The physical-chemical properties of ring substituted anilines are summarized in Table 1.

Table la. Physical-Chemical Properties of the Ring Substituted Anilines1

Property

Benzenamine,
2,6-diethyl-

Benzenamine,
2-ethyl-

Benzenamine
(supporting
chemical)

Benzenamine, 2-
ethyl-6-methyl-
(supporting
chemical)

CASRN

579-66-8

578-54-1

62-53-3

24549-06-2

Molecular Weight

149.24

121.18

93.13

135.21

Physical State

Liquid

Liquid

Liquid

Liquid

Melting Point

3.5°C (measured)

-46.5°C
(measured)

-6.2°C (measured)

-25°C (measured)

Boiling Point

235.5-242°C
(measured)

209.7-214°C
(measured)

184°C
(measured)

231°C
(measured)

Vapor Pressure

3.83xl0"3 mm Hg
at 25°C
(measured)

0.17 mm Hg at
25°C (measured)

0.49 mm Hg at
25°C (measured)

0.05-0.06 mm Hg
at 20°C (measured)

Dissociation
Constant

pKb = 9.71
(measured)2

pKb = 9.7
(measured)

pKb = 9.4
(measured)

pKb = 10.1
(estimated)3

Henry's Law
Constant

l.lxlO"6

"3

atm-m /mol
(estimated)4

6.8xl0"6

"3

atm-m /mol
(estimated)4

2.02xl0"6

"3

atm-m /mol
(measured)5

4.0xl0"6

"3

atm-m /mol
(estimated)4

5

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Water Solubility

670 mg/L at
26.7°C (measured)

4,000 mg/L at
20°C (measured)

36,000 mg/L at
20°C (measured)

2,660 mg/L at 22°C
(measured)

Log Kow

0.95 (measured)6;
3.15 (estimated)4

2.04 (measured)

0.91 (measured)

2.66 (estimated)4

1 Albemarle Corporation. 2009. Transmittal Letter and Revisions for the Ring Substituted Anilines Category.
Available online at http://www.epa.gov/hpv/pubs/summaries/ringsact/cl6251tc.htm as of August 22, 2012.
2Liu SY; Minard RD; Bollag JM. 1987. Soil-catalyzed complexation of the pollutant 2,6-diethylaniline with
syringic acid. J Environ Qual 16(l):48-54.

3SPARC. 2011. pKa/property server. Ver 4.6. Available online at http://archemcalc.com/sparc as of August 22.
2012.

4U.S. EPA. 2012. Estimation Programs Interface Suite™ for Microsoft® Windows, v4.10. U.S. Environmental
Protection Agency, Washington, DC, USA. Available online at
http://www.epa.gov/opptintr/exposure/pubs/episuitedl.htm as of August 22, 2012.

5SRC. 2012. The Physical Properties Database (PHYSPROP). SRC: Syracuse, NY. Available online at
http://www.srcinc.com/what-we-do/free-demos.aspx as of August 22, 2012.

6 This is the measured value as reported in the robust summary obtained from a published study (Lu PY, Metcalf
RL. 1975. Environ. Health Perspect.; 10: 269-84). Comparing this log Kow to the other log Kow values in this
category, suggests that the log Kow of 0.95 for benzenamine, 2,6-diethyl-is inaccurate.

2. General Information on Exposure

2.1 Production Volume and Use

The Ring Substituted Anilines Category chemicals had an aggregated production and/or import
volume in the United States between 10 to 50 million pounds during calendar year 2005.

CASRN 579-66-8: 10 to < 50 million pounds;

CASRN 578-54-1 was not reported in the 2006 IUR.

CASRN 579-66-8: Non-confidential information in the IUR indicated that the industrial
processing and uses of the chemical include pesticide and other agricultural chemical
manufacturing as intermediates. No commercial and consumer uses were reported for the
chemical.

2.2 Environmental Exposure and Fate

The ring substituted anilines are expected to have moderate mobility in soil. Benzenamine, 2,6-
diethyl- did not degrade in 14 days in pond water and was only degraded 3% after 14 days when
the pond water was amended with sewage sludge. However, it was rapidly transformed using a
non sterilized Hagerstown silt loam in soil/water slurries. More than 40 and 75% of the initially
applied amount disappeared after 5 and 20 hours, respectively. The degradation rate was shown
to be pH-dependent with >85% loss after 10 hours when the pH was adjusted to 3.8, but only
20% degradation after 10 hours at pH 7. The degradation mechanism was unclear since some
benzenamine, 2,6-diethyl- was degraded in gamma irradiated and autoclaved soils as well.
Benzenamine, 2,6-diethyl- was degraded 17-32%> after 30 days when incubated at 22°C under
anaerobic conditions using an agricultural silt loam in soil/water slurries. No degradation was

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Hazard Characterization Document

observed using a sandy loam previously exposed to metolachlor or in bovine rumen after 30 days
under anaerobic conditions. Supporting chemical benzenamine, 2-ethyl-6-methyl- achieved 0%
of its theoretical biochemical oxygen demand (BOD) after 30 days using an adapted sewage
sludge as inoculum and the closed bottle (OECD 301D) test. It was also <10% degraded after 28
days as measured by the modified OECD screening (OECD 301E) test. Supporting substance
benzenamine was shown to be readily biodegradable using the modified MITI (OECD 301C) test
and is a benchmark chemical often used as a control for aerobic ready biodegradation tests.
Volatilization is expected to be moderate for these substances. However, they will partially exist
in their protonated form (conjugate acid) rather than as the free base at environmental pH, and
the cations will not volatilize. The rate of hydrolysis is negligible. A bioconcentration factor of
120 was reported for benzenamine, 2,6-diethyl- using gambusia affinis (western mosquitofish).
The rate of atmospheric photooxidation is rapid. The ring substituted anilines are expected to
have overall moderate persistence (P2) and may be more persistent (P3) in some environmental
compartments, and low bioaccumulation potential (Bl).

The environmental fate characteristics of ring substituted anilines are summarized in Table 2.

Table lb. Environmental Fate Characteristics of the Ring Substituted Anilines1

Property

Benzenamine,
2,6-diethyl-

Benzenamine,
2-ethyl-

Benzenamine

(supporting
chemical)

Benzenamine, 2-
ethyl-6-methyl-
(supporting
chemical)

CASRN

579-66-8

578-54-1

62-53-3

24549-06-2

Photodegradati on
Half-life

0.8 hours
(estimated)

1.0 hours
(estimated)

1.2 hours
(estimated)

0.8 hours
(estimated)

Hydrolysis Half-
life

Stable

Stable

Stable

Stable

Biodegradation

0% after 14 days
in pond water;
40% after 5 hours
and 75%) after 20
hours in soil/water
slurry3;

17-32% after 30
days under
anaerobic
conditions3

No data

90% after 14 days
in pond water;
85-99% after 14
days (readily
biodegradable,
OECD 301C)4

0%> after 30 days
(not readily
biodegradable,
OECD 301D);
<10%> after 28 days
(not readily
biodegradable,
OECD 301E);
0-27% after 30
days under
anaerobic
conditions3

Bioaccumulation
Factor

BCF = 120
(measured in
mosquitofish);
BAF = 53.5

2

(estimated)

BAF = 9.8
(estimated)

BAF = 1.6

2

(estimated)

BAF = 26.8
(estimated)

7

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Table lb. Environmental Fate Characteristics of the Ring Substituted Anilines1







Benzenamine

Benzenamine, 2-









ethyl-6-methyl-



Benzenamine,

Benzenamine,

(supporting

(supporting

Property

2,6-diethyl-

2-ethyl-

chemical)

chemical)

Log Koc

2.8 (estimated)2

2.3 (estimated)2

1.9 (estimated)2

2.6 (estimated)2

Fugacity









(Level III Model)2









Air (%)

<0.1

0.1

0.3

<0.1

Water (%)

18.8

20.9

29.5

19.8

Soil (%)

80.4

78.7

70.1

79.7

Sediment (%)

0.7

0.3

0.1

0.4

Persistence5

P2-P3 (moderate

P2 (moderate to

PI (low-moderate)

P2 (moderate-high)



to high)

high)





Bioaccumulation5

Bl (low)

Bl (low)

Bl (low)

Bl (low)

1 Albemarle Corporation. 2009. Transmittal Letter and Revisions for the Ring Substituted Anilines Category.
Available online at http://www.epa.gov/hpv/pubs/summaries/ringsact/cl6251tc.htm as of August 22, 2012.
2U.S. EPA. 2012. Estimation Programs Interface Suite™ for Microsoft® Windows, v4.10. U.S. Environmental
Protection Agency, Washington, DC, USA. Available online at
http://www.epa.gov/opptintr/exposure/pubs/episuitedl.htm as of August 22, 2012.

3HSDB. 2012. Hazardous Substance Data Bank. Available online at http://toxnet.nlm.nih.gov/cgi-
bin/sis/htmlgen?HSDB as of August 22, 2012.

4Chemical Risk Information Platform (CHRIP) database. Searchable by CASRN online at
http://www.safe.nite. go. jp/english/db.html as of August 22, 2012.

5Federal Register. 1999. Category for Persistent, Bioaccumulative, and Toxic New Chemical Substances. Federal
Register 64, Number 213, pp. 60194-60204.

Conclusion: The ring substituted anilines are composed of two substances: benzenamine, 2,6-
diethyl- and benzenamine, 2-ethyl-. Benzenamine, 2,6-diethyl- is a liquid with low vapor
pressure and moderate water solubility, while benzenamine, 2-ethyl- is a liquid with low vapor
pressure and high water solubility. Both substances are expected to have moderate mobility in
soil. A supporting chemical, benzenamine, 2-ethyl-6-methyl- was not readily biodegradable in
two standard OECD screening tests and benzenamine, 2,6-diethyl- did not degrade in 14 days in
pond water, but was transformed in soil/water slurries. Volatilization is expected to be moderate
for these substances. However, they will partially exist in their protonated form (conjugate acid)
rather than as the free base at environmental pH, and the cations will not volatilize. The rate of
hydrolysis is negligible. The rate of atmospheric photooxidation is rapid. Benzenamine, 2,6-
diethyl- and benzenamine, 2-ethyl-are expected to have moderate to high persistence (P2-P3) and
low bioaccumulation potential (Bl). This assessment is based on submitted information and
limited database and literature review. A detailed review of the published literature may reveal
additional fate information.

8

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3. Human Health Hazard

A summary of health effects data for SIDS and other endpoints is provided in Table 3. The table
also indicates where available data for one chemical are read-across (RA) to the other chemical
in the category.

Acute Oral Toxicity

2, 6-Diethylaniline (CASRN 579-66-8)

(1)	Sprague-Dawley rats (15/sex/dose) were administered 2,6-diethylaniline (>98% purity) via
gavage (assumed route) at 500, 750, 1000, 1500 2000 and 2500 mg/kg. The observation period
is not indicated in the robust summary. Mortality occurred within 3 days post dosing with an
isolated delayed mortality on day 8 (details of mortality per dose are not indicated in the robust
summary.)

LD50 = 1800 mg/kg

(2)	CD rats (5 males/dose) were administered 2,6-diethylaniline (>98% purity) via gavage
(assumed route) at 1350, 2020, 3040 or 4560 mg/kg. Observation period is not indicated in the
robust summary. All animals died at 4560 mg/kg; no animals died at 1350 mg/kg. At 2020 and
3040 mg/kg each, 2/5 animals died.

LD50 = 2690 mg/kg

2-Ethylaniline (CASRN 578-54-1)

CD rats (5/sex/dose) were administered 2-ethylaniline (technical grade) via gavage at 290, 590,
1170, 2350 or 4700 mg/kg and observed for 14 days. Only five males were dosed at 4700
mg/kg. Mortalities were 0/10, 0/10, 7/10, 10/10 and 5/5 at 290, 590, 1170, 2350 and 4700
mg/kg, respectively.

LD50 = 1010 mg/kg

Acute Inhalation Toxicity
2, 6-Diethylaniline (CASRN 579-66-8)

Male rats (strain and number per concentration not specified) were exposed to 2, 6-dithey aniline
(>98%) purity) at 0, 210, 220 or 4700 mg/m3 (0, 0.21, 0.22 or 4.7 mg/L, respectively) for 1, 4 or 6
hours. The observation period is not identified in the robust summary. Rats exposed to 0.21
mg/L survived single one- and four-h exposures and 16 six-h exposures. All rats exposed to 4.7
mg/L for a single six-hour exposure died within 3 days. No information is provided on the
effects of exposure to 0.22 mg/L.

LC50 (males) > 0.22 < 4.7 mg/L

2-Ethylaniline (CASRN 578-54-1)

CD Rats (5 males) were exposed to 2-ethylaniline (technical grade) as a vapor at 1.07 mg/L for 1
or 4 hours and observed for 14 days following dosing. No mortalities were observed.
LC50 (males) > 1.07 mg/L

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Acute Dermal Toxicity
2,6-Diethylaniline (CASRN 579-66-8)

New Zealand White rabbits (4/sex/dose) were administered 2,6-diethylaniline (>98% purity) via
the dermal route on to two intact and two abraded skin sites at doses ranging from 262 to 6545
mg/kg , in contact with skin for 24 hours (conditions for occlusion are not provided).

Observation period is not indicated in the robust summary. All animals died at 2992 and 6545
mg/kg within 7 days. No deaths occurred at 262 or 393 mg/kg. Additionally, one animal each
was administered 2, 6-diethylaniline at 9350, 14,960, 22,440 or 33,660 mg/kg and mortalities
were observed at all doses.

LD50 = 1160 mg/kg

2-Ethylaniline (CASRN 578-54-1)

New Zealand White rabbits (4/sex/dose) were administered 2-ethylaniline (technical grade) via
the dermal route on to two intact and two abraded skin sites at 350, 540, 830 and 1300 mg/kg for
24 hours under occluded conditions. Observation period is not indicated in the robust summary.
All rabbits died at 1300 mg/kg on day 1 or 2 post dosing; two rabbits with abraded skin dosed at
830 mg/kg died on day 8 post dosing. No rabbits died at 540 mg/kg.

LD50 = 840 mg/kg

Repeated-Dose Toxicity
2,6-Diethylaniline (CASRN 579-66-8)

Oral

In a 90-day repeated-dose toxicity study, Sprague-Dawley rats (25/sex/dose, except 10/sex in
high-dose group) were administered 2,6-diethylaniline (>98% purity) in the diet at 0, 0.03, 0.12,
0.48 or 0.72%) (~ 0, 16.5, 66, 264 or 396 mg/kg-bw/day) for 90 days. An additional 10 males
and 10 females in 0.48%> and control groups were included for 45-day sacrifice and the 0.12%
group was included for pathological examination only. At the 45-day sacrifice, animals treated
at 0.48%o diet had slightly heavier livers and slightly elevated blood cholesterol. No effects were
during macroscopic or microscopic of organs were noted in sacrificed animals. No deaths or
altered clinical signs were observed in any treatment group. Retarded rates of body weight gain
and reduced food consumption were observed in females at days 45 and 90; but not in males. At
the 90-day sacrifice, treatment-related effects included mildly elevated serum cholesterol levels
in all animals and decreased liver weights in animals treated at 0.12%. No other notable effects
were observed in hematology, blood chemistry, urinalyses, or during microscopic examination.
LOAEL = 16.5 mg/kg-bw/day (based on increased serum cholesterol and reduced liver
weights)

NOAEL = Not established

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Dermal

In a repeated-dose dermal toxicity study, Sprague-Dawley rats (10/sex/group) were administered
2,6-diethylaniline (>98.5% purity) on to the shaved back skin of rats at 0, 100, 300 or 1000
mg/kg-day for 6 hours/day, 5 days/week for 28 days. Rats were fitted with collars to prevent
2,6-diethylaniline ingestion. After 6 hours of exposure each day, the remaining test substance at
the application site was wiped clean. Thirty tissue types were examined histologically from the
1000 mg/kg-day and control animals, and skin, kidneys and spleens were also examined from
animals dosed at 100 or 300 mg/kg-day. Three animals died at 1000 mg/kg-. Treatment-related
effects included moderately decreased body weight gain in males at 1000 mg/kg-day and a lesser
decrease in body weight gain in males at 300 mg/kg-day and in females at 1000 mg/kg-day.
Treatment-related effects included increased reticulocytes in males and females dosed at 1000
mg/kg-day and slightly increased serum glutamic pyruvic transaminase (SGPT) and/or serum
glutamic oxaloacetic transaminase (SGOT) levels (associated with slight increases in liver
weights) in animals at 300 or 1000 mg/kg/day. Treatment-related skin lesions (superficial
inflammation) and very slight histopathological lesions of the spleen (females, increased
hemosiderin) and kidneys (males, increased eosinophilic hyaline droplet formation in cytoplasm
of cortical renal tubular epithelial cells) were observed at 100 mg/kg-day. Additional treatment-
related effects observed at 300 and 1000 mg/kg-day included decreased weight gain, slightly
increased liver weights and increased serum enzymes.

LOAEL = 300 mg/kg-day (based on decreased weight gain, increased liver weights and
increased serum enzymes levels)

NOAEL = 100 mg/kg-day

Reproductive Toxicity
2,6-Diethylaniline (CASRN 579-66-8)

No reproductive toxicity data are available for 2,6-diethylaniline and 2-ethylaniline or the
supporting chemicals. Since in repeated-dose toxicity studies, described above, no effects on
reproductive organs were seen and available developmental toxicity study for 2,6-diethylaniline
addresses the reproductive toxicity endpoint.

2,6-Diethylaniline (CASRN579-66-8)

(1) In a 90-day dietary study of 2,6-diethylaniline in rats, histological examinations of tissues
from the control group and the highest two dose groups (-264 and 398 mg/kg-bw/day) at interim
(day 45) and terminal sacrifices showed no treatment-related effects on the reproductive organs
from males or females.

No effects on reproductive organ histology were seen in this study.

(2) In a 28-day dermal study of 2,6-diethylaniline in rats, 30 tissues were examined histologically
from control and high-dose animals, and no effects on reproductive organs were noted.

No effects on reproductive organ histology were noted in this study.

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Developmental Toxicity
2,6-Diethylaniline (CASRN 579-66-8)

In a prenatal developmental toxicity study, pregnant Sprague-Dawley rats (24/dose in control,
low and mid-dose groups and 29 in the high-dose group) were administered 2, 6-diethylaniline
(>98% purity) at 0, 50, 250 and 500 mg/kg-day daily via gavage during gestation days 6 through
15. Animals were observed fir mortality, body weight, food consumption and any toxicological
effects. All dams were sacrificed on day 20 and gross postmortem examinations were
conducted. Two females at 500 mg/kg-day died and here were sacrificed moribund during the
study. Salivation, anogenital staining and decreased maternal body weight gains were noted at
all doses; not significant at 50 mg/kg-day. Pregnancy rates and uterine implantations were
comparable among treated and control groups. Mean fetal body weights were comparable in
control, 50, and 250 mg/kg-day group pups. At 500 mg/kg-day, pup weights were 9% lower that
the controls. No treatment-related effects were observed regarding external, visceral or skeletal
malformations except an increased incidence of unossified sternebrae (5th and 6th vertebrae) in
500 mg/kg-day group pups.

LOAEL (maternal toxicity) = 50 mg/kg-day (based on salivation, anogenital staining and
decreased maternal body weight gains)

NOAEL (maternal toxicity) = Not established

LOAEL (developmental toxicity) = 500 mg/kg-day (based on decreased fetal weight and
increased incidence of unossified sternebrae)

NOAEL (developmental toxicity) = 250 mg/kg-day

Genetic Toxicity — Gene Mutations
In vitro

2,6-Diethylaniline (CASRN 579-66-8)

(1)	In Ames test, Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538
were exposed to 2,6-diethylaniline (98.2% purity, 1% v/v solution in dimethylsulfoxide
[DMSO]) at 0.01, 0.05, 0.10, 0.50 or 1.0 |iL/platein the presence and absence of metabolic
activation. The cytotoxic concentration was > 1.0 |iL/plate. The results were negative. The
information on the use and response of positive controls was not provided.

2,6-Diethylaniline was not mutagenic in this assay.

(2)	In a point mutation assay, Chinese hamster ovary (CHO) cells were exposed to 2,6-
diethylaniline (>98% purity) at concentrations of 300 - 500 |ag/mL in the absence and 200 - 400
|ig/mL in the presence of metabolic activation. Slightly elevated mutation frequencies were seen
at several concentrations producing significant cytotoxicity, but these increases were weak, and
not statistically significant. Six additional concentrations, ranging from 450 to 475 |ig/mL and
275-400 |ig/mL, in absence and presence of metabolic activation, respectively, were tested to
clarify inconsistent results. Only weak and sporadic increases in mutation frequency were noted
following incubation of Chinese hamster ovary cells with 2,6 diethylaniline. These increases
were neither statistically significant nor dose related. Therefore, 2,6-diethylaniline is not
considered to be mutagenic at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT)
locus of Chinese hamster ovary cells.

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2,6-Diethylaniline was not mutagenic in this assay.

(3) Salmonella typhimurium strain TA100 was exposed to 2,6-diethylaniline (98.27% purity) at
concentrations ranging from 0.003 to 3.00 |iL/plate in the presence and 0.0015 to 2.25 |iL/plate
in the absence of metabolic activation. Very slight but apparently dose-related increases in
revertants were observed in glass and plastic petri dishes with activation. In the absence of
activation, a slight increase in revertants was seen in plastic but not glass dishes. Responses
were never more than twice those in solvent controls, and not always reproducible. Conclusion
was that 2,6 diethylaniline was weakly mutagenic in TA 100 in the presence of activation, but
the low level of response was not considered to be biologically significant. No information was
provided on the use and response of the positive controls.

2,6-Diethylaniline was weakly mutagenic in this assay.

(4) Salmonella typhimurium strains TA98 and TA100 were exposed to 2,6-diethylaniline (>98%
purity) at concentrations ranging from 0.25 to 3000 |ig/plate with and without metabolic
activation. Cytotoxic concentration was >1 mg/plate without activation and 3 mg/plate and
above with metabolic activation. The test substance produced negative results in TA98 with and
without activation and weakly mutagenic response in TA100 in the presence of activation. No
information was provided on the use and response of the positive controls.

2,6-Diethylaniline was weakly mutagenic in this assay.

(5) In Ames test, Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 were
exposed to 2,6-diethylaniline (99.5% purity) at 0, 0.05, 0.15, 1.50 or 5.0 mg/plate with and
without metabolic activation. Cytotoxicity was observed at 5.0 mg/plate. For TA 100 and TA
1535, observed increases in revertants/plate were limited to 1.5 to 2 fold over control values,
generally at 1.5 mg/plate. Although there was some activity in the absence of S-9, more
consistent activity was observed in the presence of nasal turbinate S-9. There were no substantial
differences between rat, mouse or monkey S-9, but the responses were marginal, limiting the
comparison.

Positive and negative controls were used, but their response was not provided. No information
was provided with regards to observed precipitation.

2,6-Diethylaniline was equivocal for mutagenicity in this assay.

2-Ethylaniline (CASRN 578-54-1)

Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 were exposed to
2-ethylaniline (99.6% purity) at 0, 0.05, 0.15, 0.5, 1.5 or 5.0 without and at 0.15, 0.5, 1.5, 5.0 or
15 |iL/mL with metabolic activation.. Solvent and positive controls were used; however, no
information was provided on their response. No significant increase in revertants was noted at
any concentration either with and without metabolic activation..

2-Ethylaniline was not mutagenic in this assay.

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Genetic Toxicity - Chromosomal Aberrations
In vitro
No data
In vivo

2,6-Diethylaniline (CASRN 579-66-8)

In a micronucleus test, BS-1 mice (4/sex/group) were administered 2,6-diethylaniline (98.2%
purity) in 0.25% methylcellulose via intraperitoneal injection at 100 or 200 mg/kg as a split-dose
separated by 24 hours. Polychromatic erythrocytes from bone marrow extractions were
examined for micronuclei. Mice showed signs of systemic toxicity (increased spontaneous
activity, mild convulsions, ataxia and/or loss of righting). Negative and positive controls were
included and responded appropriately. There was no increase in the number of micronuclei in
polychromatic erythrocytes

2,6-Diethylaniline did not induce micronuclei in mice in this assay.

Additional Information
Skin Irritation

2,6-Diethylaniline (CASRN 579-66-8)

Rabbits (six animals; sex and strain not specified) were administered 0.7 g 2,6-diethylaniline
(>98%) purity) dermally on to the intact and abraded skin at 0.7 g for 24 hours under semi-
occlusive conditions. Scoring was made for Erythema, edema and eschar at 24 and 72 hours
following dosing. Erythema and edema were observed on the intact sites of three animals at 24
hours and one animal at 72 hours. The Primary Irritation Index was 2.7.

2,6-Diethylaniline was slightly irritating to rabbit skin in this study.

2-Ethylaniline (CASRN 578-54-1)

Six rabbits (sex and strain not specified) were administered 0.5 mL 2-ethylaniline (technical
grade) dermally on to the intact and abraded skin sites for 24 hours under semi-occlusive
conditions Scoring for Erythema, edema and eschar was made at 24 and 72 hours following
dosing. The Primary Irritation Score was 0.33. Two animals had erythema score of "1" at 24
hour reading while others had a score of "0".

2-Ethylaniline was not irritating to rabbit skin in this study.

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Eye Irritation

2,6-Diethylaniline (CASRN 579-66-8)

Rabbits (seven animals; sex and strain not specified) received an ocular administration of 2,6-
diethylaniline (>98% purity, amount not specified) and were observed for 10 days following
instillation. Treatment-related effects included swelling and erythema in the tissues around the
eyes, cloudiness of the cornea and purulent discharge. Microscopic examination of the eyes
showed marked edema with infiltration by lymphocytes and polymorphonuclear leucocytes
within the eyelids. Conjunctivae were markedly hyperemic. Minute ulcerations were present in
the cornea.

2,6-Diethylaniline was irritating to rabbit eyes in this study.

2-Ethylaniline (CASRN 578-54-1)

Six rabbits (males and females; strain not specified) had instilled 0.1 mL of 2-ethylaniline
(technical grade) in one eye and were observed for > 7 days following instillation. All rabbits
showed slight irritation of corneas and conjunctivae after 24 hours. All treated eyes showed
improvement by day 7.

2-Ethylaniline was mildly irritating to rabbit eyes in this study.

Sensitization

2,6-Diethylaniline (CASRN 579-66-8)

Guinea pigs (six test and four positive control animals; sex and strain not specified) were
exposed to 2,6-diethylaniline (>98% purity) in an induction phase consisting of a 0.05 mL
intracutaneous injection followed by nine 0.1 mL intracutaneous injections given on every other
day. Two weeks after the 10th injection, a 0.05 mL intracutaneous challenge injection was made.
Positive control animals were administered 0.1% dinitrochlorobenzene and responded
appropriately. None of the animals reacted to sensitizing to challenge injections.
2,6-Diethylaniline was not sensitizing to guinea pigs in this study.

Carcinogenicity

2,6-Diethylaniline (CASRN 579-66-8)

Sprague-Dawley rats (80/sex/concentration, 100/sex control group) were administered 2,6-
diethylaniline (>98%) purity) in the diet daily at 0, 0.02, 0.16 or 0.32% for 104 weeks. Clinical
chemistry and hematological evaluations included methemoglobin analysis. Urine analysis was
also conducted. Histopathological examination was conducted on gross lesions and a standard
set of tissues. Reproductive organs evaluated included testes, prostates, epididymides, seminal
vesicles, Fallopian tubes, ovaries, uteruses, cervixes, mammary glands and vaginas. No effects
were observed on survival, food consumption, clinical pathology values, organ weights or
incidence of neoplastic or non-neoplastic lesions. A statistically significant increase in number
of female rats exhibiting adenocarcinomas of the mammary gland was observed in the high dose
animals compared to control. However, the incidence was similar to that reported to occur

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spontaneously in female Sprague Dawley rats. There was not an increase in the number of
neoplasms per animal. Retardation of body weight gain was observed in high-dose male and
female rats at each 6-month interval.

2,6-Diethylaniline did not increase tumor incidence in this study.

Conclusion: Acute oral toxicity of 2,6-diethylaniline and 2-ethylaniline in rats is low; acute
inhalation toxicity in rats is moderate and acute dermal toxicity in rabbits is moderate. In a 90-
day dietary repeated-dose toxicity study of 2,6-diethylaniline in rats, increased cholesterol and
decreased liver weights were noted >16.5 mg/kg-bw/day. The NOAEL is not established. In the
28-day repeated-dose dermal toxicity study of 2,6-diethylaniline in rats, decreased body weight
gain, increased liver weights and increased serum enzyme levels (serum glutamic oxaloacetate
transaminase, (SGOT) and serum glutamic pyruvic transaminase (SGPT)) were observed at
> 300 mg/kg-day. The NOAEL is 100 mg/kg-day.

2,6-Diethylaniline was mutagenic showing weak response but 2-Ethylaniline was not mutagenic
in bacteria in vitro. 2,6-Diethylaniline did not induce micronuclei in mice
in vivo. 2,6-Diethylaniline was irritating to rabbit skin and irritating to rabbit eyes.

2-Ethylaniline was not irritating to rabbit skin; but was irritating to rabbit eyes.

2,6-Diethyaniline is not sensitizing to guinea pigs. 2,6-Diethylaniline did not increase tumor
incidence in rats.

Table 3. Summary of the Screening Information Data Set as Submitted under the U.S.
HPV Challenge Program -Human Health Data

Endpoints

2,6-Diethylaniline
(579-66-8)

2-Ethylaniline
(578-54-1)

Acute Oral Toxicity
LD5o (mg/kg)

1800
2690

1010

Acute Dermal Toxicity
LD50 (mg/kg)

1160

840

Acute Inhalation Toxicity
LC50 (mg/L)

>0.22 < 4.7

>1.07

Repeated-Dose Toxicity

NOAEL/LOAEL

Oral

(90-day, diet)
NOAEL = Not established
LOAEL = 16.5

No data
NOAEL = Not established
LOAEL = 16.5

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(mg/kg-bw/day)



(RA)

Repeated-Dose Toxicity
NOAEL/LOAEL
Dermal
(mg/kg-day)

(28-day)
NOAEL = 100
LOAEL = 300

No data
NOAEL = 100
LOAEL = 300
(RA)

Reproductive Toxicity
NOAEL/LOAEL
Oral (mg/kg-bw/day)

No effects were seen following

evaluation of reproductive
organs in 28-day dermal and
90-day oral toxicity studies in
rats.

No data
No effects were seen
following evaluation of
reproductive organs in 28-
day dermal and 90-day oral
toxicity studies in rats.
(RA)

Developmental Toxicity
NOAEL/LOAEL
Oral (mg/kg-bw/day)

Maternal Toxicity

NOAEL = Not established
LOAEL = 50

No data
Not established
50

Developmental Toxicity

NOAEL = 250
LOAEL = 500

NOAEL = 250
LOAEL = 500
(RA)

Genetic Toxicity - Gene
Mutation- in vitro

Weakly positive

Negative

Genetic Toxicity -
Chromosomal Aberration
in vitro

No data

Nodata

Genetic Toxicity -
Chromosomal Aberration

in vivo

Negative

No data
Negative
(RA)

Other Information

Skin Irritation
Eye Irritation
Sensitization
Carcinogenicity

Irritating
Irritating
Negative
Did not increase tumor
incidence

Not irritating
Irritating

No data

Measured data in bold text; (RA) = Read Across; - indicates that endpoint was not evaluated for this compound;

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4. Hazard to the Environment

A summary of ecotoxicity data for SIDS and other endpoints is provided in Table 4. The table
also indicates where data for the supporting chemical are read-across (RA) to the sponsored
substances.

Acute Toxicity to Fish
2,6-Diethylaniline (CASRN 579-66-8)

(1)	Rainbow trout (iOncorhynchus mykiss) were exposed to 2,6-diethylaniline (98.8% purity) at
unspecified concentrations under static conditions for 96 hours.

96-h LC50 = 24 mg/L

(2)	Bluegill sunfish (Lepomis macrochirus) were exposed to 2,6-diethylaniline (98.8% purity) at
unspecified concentrations under static conditions for 96 hours.

96-h LC50 = 30 mg/L

2-Ethylaniline (CASRN 578-54-1)

Guppies (Poecilia reticulata) were exposed to 2-ethylaniline (> 98% purity) prepared in
isopropanol at unspecified concentrations under static-renewal conditions for 14 days.
14-d LC50= 74.7 mg/L

Acute Toxicity to Aquatic Invertebrates
2-Ethylaniline (CASRN 578-54-1)

Water fleas (Daphnia magna) were exposed to 2-ethylaniline (> 98% purity) prepared in
isopropanol at unspecified concentrations under static conditions for 48 hours. The 48-h LC50
value of 12 mg/L was predicted by ECOSAR v. 1.11 to support the evaluation of aquatic
invertebrate toxicity.

48-h EC50 = 8 mg/L

2,6-Diethylaniline (CASRN 579-66-8)

Water fleas (D. magna) were exposed to 2,6-diethylaniline (98.8% purity) at unspecified
concentrations under static conditions for 48 hours.

48-h EC50 = 21 mg/L

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Toxicity to Aquatic Plants
2-Ethylaniline (CASRN 578-54-1)

Green algae (Chlorellapyrenoidosa) were exposed to 2-ethylaniline (> 98% purity) prepared in
isopropanol at unspecified concentrations under static conditions for 96 hours. The 96-h EC50
value of 41 mg/L was predicted by ECOSAR v. 1.11 to support the evaluation of aquatic plant
toxicity.

96-h EC50 (growth rate) = 38 mg/L

Chronic Toxicity to Aquatic Invertebrates
2-Ethylaniline (CASRN 578-54-1)

Water fleas (D. magna; 10 replicates of 1 daphnid per test concentration) were exposed to 2-
ethylaniline at unspecified concentrations under static-renewal conditions for 21 days. The
lowest rejected concentration tested (LRCT) that significantly lowered the population intrinsic
growth rate (rm) of the daphnids was 1 mg/L and the LRCT that significantly lowered the mean
length of daphnids after 21 days of exposure was 3.2 mg/L.

21-d LC50 = 7 mg/L

Conclusion: For the ring-substituted anilines category members, the 96-hour LC50 for fish is 24
mg/L, the 48-h EC50 for aquatic invertebrates ranges from 8 to 21 mg/L and the 96-h EC50 for
aquatic plants is 38 mg/L for growth rate.

Table 4. Summary of the Screening Information Data Set as
Submitted under the U.S. HPV Challenge Program- Aquatic toxicity

Endpoints

2,6-Diethylaniline
(579-66-8)

2-Ethylaniline
(578-54-1)

Fish

96-h LC50 (mg/L)

24

No Adequate Data
24

(RA)

Aquatic Invertebrates
48-h EC50 (mg/L)

21

8

Aquatic Plants
96-h EC50 (mg/L)

(growth rate)

No Data
38
(RA)

38

(Bold) = experimental data (i.e., derived from testing); (RA) = Read Across

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