Registration Dossier

Administrative data

Description of key information

Rat studies:

28-Day rat feeding study LOAEL: 20000/10000 ppm (equivalent to 653 mg/kg bw/day) for males and 4000 ppm (equivalent to 317 mg/kg bw/day) for females; OECD 407; Reliability = 1

90-Day rat feeding study LOAEL: 6000 ppm (equivalent to 274 mg/kg bw/day and 316 mg/kg bw/day for males and females respectively); OECD 408; Reliability = 1

90-Day rat feeding study LOAEL: 6000 ppm (equivalent to 257 mg/kg bw/day and 278 mg/kg bw/day for males and females respectively); OECD 408; Reliability = 1

29-Day rat dermal study LOAEL: > 1000 mg/kg; OECD 410; Reliability = 1

Mouse studies:

28-Day mouse feeding study LOAEL: > 7000 ppm (equivalent to 1104 mg/kg bw/day and 1343mg/kg bw/day for males and females respectively); OECD 407; Reliability = 1

90-Day mouse feeding study LOAEL: > 7000 ppm (equivalent to 274 mg/kg bw/day and 316 mg/kg bw/day for males and females respectively); OECD 408; Reliability = 1

Dog studies:

28-Day dog feeding study LOAEL: 3000 ppm (equivalent to 49.52 mg/kg bw/day and 67.07 mg/kg bw/day for males and females respectively); no guideline; Reliability = 1

90-Day dog feeding study LOAEL: 1000 ppm (equivalent to 26.6 mg/kg bw/day and 26.87 mg/kg bw/day for males and females respectively); OECD 409; Reliability = 1

12-Month dog feeding study LOAEL: > 2000 ppm (equivalent to 53.18 mg/kg bw/day and 55.88 mg/kg bw/day for males and females respectively); OECD 452; Reliability = 1

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
Deviations:
yes
Qualifier:
according to
Guideline:
other: US EPA 870.4200
Deviations:
yes
Qualifier:
according to
Guideline:
EU Method B.30 (Chronic Toxicity Studies)
Deviations:
yes
Qualifier:
according to
Guideline:
other: Ministry of Agriculture, Forestry and Fisheries in Japan, Guidelines for Data Requirements for Supporting Registration of Pesticides, No. 12-Nousan-No.8147, Notification by Director-General dated 24 November, 2000
Deviations:
yes
GLP compliance:
yes
Species:
dog
Strain:
Beagle
Details on species / strain selection:
The beagle was selected for use in this study because the beagle is the usual non-rodent model used for evaluating the toxicity of various test articles and for which there is a large historical database.
Sex:
male/female
Route of administration:
oral: feed
Details on route of administration:
The oral route is one of the potential routes of administration of this test article in humans.
Vehicle:
other: Ground meal Lab Diet
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
52 weeks
Frequency of treatment:
Once daily (approximately 3 hours in the morning)
Dose / conc.:
40 ppm
Remarks:
(Male: 1.53 mg/kg bw/d; Female: 1.20 mg/kg bw/d)
Dose / conc.:
100 ppm
Remarks:
(Male: 3.31 mg/kg bw/d; Female: 3.37 mg/kg bw/d)
Dose / conc.:
400 ppm
Remarks:
(Male: 11.09 mg/kg bw/d; Female: 10.79 mg/kg bw/d)
Dose / conc.:
2 000 ppm
Remarks:
(Male: 53.18 mg/kg bw/d; Female: 55.88 mg/kg bw/d)
High-dose female dogs received the test article at a concentration of 1000 ppm for 13 weeks, then the concentration was increased to 2000 ppm for the remainder of the study.
No. of animals per sex per dose:
4
Control animals:
yes, plain diet
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Food efficiency:
effects observed, non-treatment-related
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Clinical biochemistry findings:
effects observed, non-treatment-related
Urinalysis findings:
effects observed, non-treatment-related
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Gross pathological findings:
effects observed, non-treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Key result
Dose descriptor:
NOAEL
Effect level:
2 000 ppm
Sex:
male/female
Basis for effect level:
other: no adverse effects noted at highest dose tested
Remarks on result:
other: 2000 ppm is equivalent to 53.18 and 55.88 mg/kg bw/day in males and females, respectively.
Critical effects observed:
no
Conclusions:
NOAEL (male/female): 2000 ppm (Highest concentration tested) (equivalent to 53.18 and 55.88 mg/kg bw/day in males and females, respectively).
Executive summary:

The study was conducted according to guidelines, US EPA OPPTS 870.4200 and OECD Guideline 452 to evaluate the toxicity of test article when administered to beagle dogs via the diet for 1 year.

Four groups of beagle dogs (4/sex/group) received the test article orally via dietary administration for one year, at dietary concentrations of 40, 100, 400, and 2000 (males) ppm. High-dose female dogs received the test article at a concentration of 1000 ppm for 13 weeks, then the concentration was increased to 2000 ppm for the remainder of the study. The control dogs (4/sex) received untreated canine diet.

Assessment of toxicity was based on mortality, clinical observations, body weight, body weight gain, food consumption, food efficiency, and compound consumption; ophthalmoscopic and physical examinations; and clinical and anatomic pathology.

The overall mean daily intake values of test substance for male dogs in the 40, 100, 400, and 2000 ppm dosing groups were 1.53, 3.31, 11.09, and 53.18 mg/kg body weight/day. The mean daily intake values for females in these dose groups (female high dose was 1000/2000 mg/kg/day) were 1.20, 3.37, 10.79, and 55.88 mg/kg body weight/day.

There were no test substance-related effects on survival, clinical findings, body weight/body weight change, nutritional parameters (food consumption and food efficiency), ophthalmoscopic and physical examinations, clinical pathology evaluations (hematology, coagulation, clinical chemistry, or urinalysis), or anatomic pathology evaluations (macroscopic, organ weight, and microscopic).

A single 400 ppm female was euthanized in extremis on Day 310 following veterinary consultations. The animal exhibited the following clinical signs prior to euthanasia: decreased activity, soft/watery feces, vomitus, and inappetence, accompanied by body weight loss. Attempts to stimulate her appetite with supplemental wet canned dog food resulted in no success. The cause of death of this female was considered incidental and related to arterial inflammation (polyarteritis), with the most prominent lesions observed in the heart, which likely contributed to morbundity. The arterial inflammation correlated with clinical pathology evidence of an inflammatory response. The lesions were considered consistent with idiopathic polyarteritis of beagle dogs, which is considered a spontaneous arterial disease primarily found in laboratory beagle dogs and of unknown etiology.

Under the conditions of this study, the no-observed-adverse-effect-level (NOAEL) for test substance was 2000 ppm in male and female dogs, the highest concentration administered (female high dose was 1000/2000 ppm). This NOAEL is equivalent to 53.18 and 55.88 mg/kg bw/day in males and females, respectively.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
yes
Qualifier:
according to
Guideline:
other: MAFF Japan, 2-1-9 Notification 12 Nousan 8147, Agricultural Chemicals Regulation Laws (2000)
Deviations:
yes
GLP compliance:
yes
Species:
mouse
Strain:
other: Crl:CD1®(ICR)
Details on species / strain selection:
The mouse is a frequently used model for evaluating the toxicity of various classes of chemicals and for which there is a large historical database.
Sex:
male/female
Route of administration:
oral: feed
Details on route of administration:
The oral route is one of the potential routes of human exposure to this test article.
Vehicle:
other: Certified Rodent Diet # 5002
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
ad libitum for 13 consecutive weeks.
Dose / conc.:
200 ppm
Remarks:
Male: 31.37 mg/kg bw/d; Female: 44.14 mg/kg bw/d
Dose / conc.:
800 ppm
Remarks:
Male: 125.44 mg/kg bw/d; Female: 177.27 mg/kg bw/d
Dose / conc.:
2 500 ppm
Remarks:
Male: 416.70 mg/kg bw/d; Female; 476.24 mg/kg bw/d
Dose / conc.:
7 000 ppm
Remarks:
Male: 1127.80 mg/kg bw/d; Female: 1526.02 mg/kg bw/d
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Clinical biochemistry findings:
effects observed, non-treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test article-related, but not adverse changes in organ weight were present in the adrenal gland and liver. Increases in absolute and relative weights of the liver in males and females at 7000 ppm and in males at 2500 ppm were attributed to test article administration; the increases were associated with increased incidence of minimal hepatocellular hypertrophy. Increases in absolute and relative weights of the adrenal glands in males and females at 7000 ppm (not statistically significant) were also attributed to test article administration but there were no microscopic correlates to the increased weights.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test article-related microscopic findings were present in the liver. Males given 2500 or 7000 ppm in the diet and females given 7000 ppm in the diet had an increased incidence of minimal hepatocellular hypertrophy which correlated with increased liver weights. The hepatocellular hypertrophy was not considered adverse because of the minimal nature of the change.
Key result
Dose descriptor:
NOAEL
Effect level:
7 000 ppm
Based on:
other: absence of adverse effects in all endpoints examined at the highest dose
Sex:
male/female
Remarks on result:
other: 7000 ppm equivalent to 1127.80 and 1526.02 mg/kg bw/d for males and females respectively.
Critical effects observed:
no
Conclusions:
NOAEL (male/female): 7000 ppm (Highest concentration tested) (Based on no adverse efffects observed at 7000 ppm in both males and females)
Executive summary:

The study was conducted according to guidelines, U.S. EPA OPPTS 870.3100 and OECD 408 to evaluate the subchronic toxicity of test substance to mice when administered in the diet for 13 weeks. Four treatment groups of ten male and ten female mice were administered the test article at respective dietary concentrations of 200, 800, 2500, and 7000 ppm. One additional group of ten animals/sex served as the control and received untreated rodent diet. The untreated or treated rodent diet was available to all groups ad libitum for 13 weeks.

Observations for morbidity, mortality, injury, and the availability of food and water were conducted twice daily for all animals. Observations for clinical signs were conducted on Day 1 and weekly thereafter for main study animals. Additional cageside observations were conducted once daily for main study animals, except for days that clinical observations were conducted. Body weights were measured and recorded predose on Day 1 and weekly thereafter for all animals. Food consumption was measured and recorded weekly, and food efficiency and compound consumption were calculated for main study animals. Ophthalmoscopic examinations were conducted on all animals (main study) pretest and on all surviving main study animals prior to the terminal necropsy. Blood samples for clinical pathology evaluations were collected from all surviving main study animals prior to the terminal necropsy. At study termination, necropsy examinations were performed and organ weights were recorded for main study animals. A full complement of tissues was microscopically examined for main study animals at 0 and 7000 ppm, with gross lesions and potential target organs (liver and kidney) examined at 200, 800, and 2500 ppm.

No test article-related effects were noted on the following parameters: survival, clinical findings, body weight/body weight gain, food consumption/food efficiency, ophthalmoscopic evaluations, hematology and clinical chemistry evaluations, or macroscopic evaluations. There was one unscheduled death each in the 0 ppm (male) and 200 ppm (female) groups; the cause of death was not determined but was not attributed to the test article.

Test article-related changes in organ weight and/or microscopic pathology were present in the adrenal gland and liver. Increases in absolute and relative weights of the liver in males and females at 7000 ppm and in males at 2500 ppm were attributed to test article administration; the increases were associated with increased incidence of minimal hepatocellular hypertrophy.

However, based on the minimal severity noted microscopically, the findings were not considered adverse and are consistent with a nonadverse increase in liver metabolizing enzymes. Increases in absolute and relative weights of the adrenal glands in males and females at 7000 ppm (not statistically significant) were also attributed to test article administration but there were no microscopic correlates to the increased weights and the increases were not considered adverse.

Under the conditions of this study, exposure to 200, 800, 2500, or 7000 ppm produced no adverse effects in either male or female mice. Therefore, the No-Observed-Adverse-Effect Level (NOAEL) for the study was considered to be 7000 ppm (1127.80 mg/kg body weight/day for males and 1526.02 mg/kg body weight/day for females).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
see "any other information on methods and methods"
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
see "any other information on methods and methods"
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
see "any other information on methods and methods"
Qualifier:
according to
Guideline:
other: MAFF Japan, 2-1-9 Notification 12 Nousan 8147, Agricultural Chemicals Regulation Laws (2000)
Deviations:
yes
Remarks:
see "any other information on methods and methods"
GLP compliance:
yes
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The rat is the rodent model routinely used for evaluating the toxicity of various classes of chemicals and for which there is a large historical database.
Sex:
male/female
Route of administration:
oral: feed
Details on route of administration:
The oral route is one of the potential routes of human exposure to this test article.
Vehicle:
other: Meal Lab Diet
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
The test article was available in the diet ad libitum.
Dose / conc.:
100 ppm
Remarks:
Males: 4.5 mg/kg bw/d; Female: 6 mg/kg bw/d
Dose / conc.:
400 ppm
Remarks:
Male: 18 mg/kg bw/d; Female: 23 mg/kg bw/d
Dose / conc.:
1 500 ppm
Remarks:
Male: 70 mg/kg bw/d; Female: 83 mg/kg bw/d
Dose / conc.:
6 000 ppm
Remarks:
Male: 274 mg/kg bw/d; Female: 316 mg/kg bw/d
No. of animals per sex per dose:
16
Control animals:
yes, plain diet
Clinical signs:
effects observed, non-treatment-related
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Adverse, test substance-related decreases (compared to control) in body weight parameters were observed in male and female rats at 6000 ppm. Overall (Weeks 1-13) mean body weight change at 6000 ppm for males and females was 21% (statistically significant) and 6% (not statistically significant), respectively, lower than control.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
-Adverse, test substancee-related decreases (compared to controls) in food intake parameters were observed in male and female rats at 6000 ppm, and correlated with the body weight effects. Statistically significant decreases in weekly mean food consumption were noted for all 13 weeks in males at 6000 ppm and for 11 of the 13 weeks in females at 6000 ppm (Week 6 and 12 values did not reach statistical significance). Statistically significant decreases in mean food consumption were noted for all 3 monthly intervals in males and females at 6000 ppm. Overall (Weeks 1-13) mean food consumption of males and females at 6000 ppm was statistically significantly lower (11% and 16%, respectively) compared to control.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
- During the first week of the study, feed efficiency values were statistically significantly decreased at 6000 ppm for both sexes when compared to control. A statistically significant decrease (21%) in the mean food efficiency for Month 2 was noted in males at 6000 ppm.
- Overall (Weeks 1-13) food efficiency of males and females at 6000 ppm were lower than controls for males (11% which is not statistically significant) and higher than controls for females (12% which is not statistically significant).
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
.
Clinical biochemistry findings:
effects observed, non-treatment-related
Urinalysis findings:
effects observed, non-treatment-related
Behaviour (functional findings):
no effects observed
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- Test substance related organ weight changes were limited to increased absolute and relative liver weights of males and females at 6000 ppm. Increased absolute liver, relative liver to body weight percentage and relative liver to brain weight ratios were observed in males by 5%, 19% (statistically significant) and 3%, and in females by 11%, 17% (statistically significant) and 9%, respectively. The increased liver weights correlated with minimal microscopic centrilobular hepatocellular hypertrophy in males at 6000 ppm and were considered most likely adaptive and non-adverse.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test substance related microscopic changes were limited to the livers of males at 6000 ppm. Minimal centrilobular hepatocellular hypertrophy was observed in 4 of 10 males at 6000 ppm. The centrilobular hypertrophy was characterized by minimal enlargement of the hepatocytes surrounding the central vein. The hepatocellular hypertrophy was considered non-adverse and consistent with an adaptive increase in liver enzyme content.
Histopathological findings: neoplastic:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
1 500 ppm
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
food efficiency
Remarks on result:
other: 1500 ppm is equivalent to 70 and 83 mg/kg body weight/day for males and females, respectively
Critical effects observed:
no
Conclusions:
NOAEL(male/female): 1500 ppm (70 and 83 mg/kg body weight/day for males and females, respectively) (Based on adverse effects on body weight and nutritional parameters in male and female rats at 6000 ppm)
Executive summary:

The test was conducted according to guidelines, U.S. EPA OPPTS 870.3100, 870.6200 and OECD Guideline 408 to evaluate the subchronic toxicity of test substance when administered in the diet of rats for 13 weeks. Four treatment groups of 16 male and 16 female rats were administered the test article at respective dietary concentrations of 100, 400, 1500, and 6000 ppm. One additional group of 16 animals/sex served as the control and received untreated rodent diet. The untreated or treated rodent diet was available to all groups ad libitum for 13 weeks.

Observations for morbidity, mortality, injury, and the availability of food and water were conducted twice daily for all animals. Additional cageside observations were conducted once daily, except on the days of the weekly detailed clinical observation. Body weights were measured and recorded prior to randomization, predose on Day 1, and weekly thereafter. Food consumption was measured and recorded weekly. Food efficiency and compound consumption were calculated weekly. Ophthalmoscopic examinations were conducted on all animals pretest and on all surviving animals prior to the terminal necropsy. Functional Observational Battery (FOB) and Motor Activity (MA) tests were conducted on designated animals (10/sex/group) pretest and during Weeks 4, 8, and 13. Blood samples for clinical pathology evaluations were collected from designated animals at the terminal necropsy. Urine samples for clinical pathology evaluations were collected from designated animals prior to the terminal necropsy. Blood samples for possible determination of the plasma concentrations of the test article and/or metabolites were collected from designated animals on Day 60. During Week 14, the last six animals/sex/group were necropsied for neuropathology evaluations. At study termination for non-neuropathology animals, necropsy examinations were performed, organ weights were recorded, and tissues were microscopically examined. A full complement of tissues was microscopically examined for animals at 0 and 6000 ppm, with gross lesions and a potential target organ (liver) examined at 100, 400, and 1500 ppm.

The overall mean compound consumption of test substance in the 100, 400, 1500, and 6000 ppm groups was 4.5, 18, 70, and 274 mg/kg body weight (bw)/day, respectively, for male rats and 6.0, 23, 83, and 316 mg/kg bw/day, respectively, for female rats.

No test substance-related effects were noted on the following parameters: survival, clinical findings, functional observational battery, locomotor activity, ophthalmoscopic evaluations, hematology, coagulation, clinical chemistry, urine/urine chemistry, or macroscopic evaluations. There was one unscheduled death in the 100 ppm (female) group on test day 88; the cause of death was undetermined but was not attributed to the test article.

Adverse, test substance-related decreases (compared to control) in body weight parameters were observed in male and female rats at 6000 ppm. Weekly mean body weight changes were also generally lower compared to controls in males and females at 6000 ppm, but with only occasional statistical significance. Overall (Weeks 1-13) mean body weight change at 6000 ppm for males and females was 21 (statistically significant) and 6% (not statistically significant), respectively, lower than control.

Adverse, test substance-related decreases (compared to controls) in food intake parameters were observed in male and female rats at 6000 ppm, and correlated with the body weight effects. Overall (Weeks 1-13) mean food consumption of males and females at 6000 ppm was statistically significantly lower (11% and 16%, respectively) compared to control. Weekly food efficiency values were generally similar to control except for Week 1, when food efficiency was statistically significantly decreased in males and females at 6000 ppm compared to control.

Test substance related organ weight changes were limited to increased absolute and relative liver weights of males and females at 6000 ppm. The increased liver weights correlated with minimal centrilobular hepatocellular hypertrophy observed in 4 of 10 males at 6000 ppm. The hepatocellular hypertrophy was considered non-adverse and likely adaptive (enzymatic induction). There were no test substance related microscopic findings consistent with neurotoxicity.

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) of test substance was considered to be 1500 ppm (70 and 83 mg/kg body weight/day for males and females, respectively). The NOAEL was based on adverse effects on body weight and nutritional parameters in male and female rats at 6000 ppm.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
other: MAFF Japan, 2-1-9 Notification 12 Nousan 8147, Agricultural Chemicals Regulation (2000)
Deviations:
no
GLP compliance:
yes
Species:
rat
Strain:
other: [Crl:CD®(SD)]
Details on species / strain selection:
The rat is the usual rodent model used for evaluating the toxicity of various classes of chemicals and for which there is a large historical database.
Sex:
male/female
Route of administration:
oral: feed
Details on route of administration:
The oral route is one of the potential routes of human exposure to this test article.
Vehicle:
other: Ground meal Lab Diet
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
ad libitum for up to 13 weeks.
Dose / conc.:
100 ppm
Remarks:
Male: 4.17 mg/kg bw/d; Female: 5.13 mg/kg bw/d
Dose / conc.:
400 ppm
Remarks:
Male: 17.01 mg/kg bw/d; Female: 20.38 mg/kg bw/d
Dose / conc.:
1 500 ppm
Remarks:
Male: 63.86 mg/kg bw/d; Female: 74.26 mg/kg bw/d
Dose / conc.:
6 000 ppm
Remarks:
Male: 257.09 mg/kg bw/d; Female: 278.14 mg/kg bw/d
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Adverse, test substane-related decreases (compared to control) in mean body weight parameters were observed in males and females at 6000 ppm. Body weights of males and females in the 6000 ppm group were statistically significantly lower compared to controls beginning on study Day 7. The statistical significance persisted for all weekly intervals for both sexes. On study Day 91 mean body weight was decreased by 11.0% and 12.3% in males and females, respectively, at 6000 ppm compared with controls.
- Weekly mean body weight changes were generally lower in both sexes at 6000 ppm compared to controls, with only occasional statistical significance. Overall (Days 1 – 91) mean body change at 6000 ppm was statistically significantly lower than controls by 20% and 32% in males and females, respectively (statistically significant).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Adverse, test substane-related decreases (compared to controls) in food intake parameters were observed in both sexes at 6000 PPM, and correlated with the body weight effects. Overall (Days 1-91) mean food consumption of both sexes at 6000 ppm was statistically significantly lower (14.4% for males and 24.2% for females) compared to controls.
- Food consumption was also decreased in females at 1500 ppm compared to controls. This decreased food consumption in females at 1500 ppm was considered test article-related but not adverse as there were no corresponding effects in body weight parameters.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
effects observed, non-treatment-related
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- Mild test article related decreases in red cell mass (erythrocytes, hemoglobin, and hematocrit) (up to -8%) and eosinophils (-61%), relative to controls. These changes were statistically significant but generally remained within historical ranges.
Urinalysis findings:
effects observed, non-treatment-related
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- The only test article-related organ weight changes consisted of minimally increased liver weights in males and females at 6000 ppm and increased uterus with cervix weights in females at 6000 ppm, which were statistically significant when expressed relative to body weight.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
1 500 ppm
Based on:
other: Based on adverse effects on body weight and nutritional parameters in male and female rats at 6000 ppm.
Sex:
male/female
Remarks on result:
other: 1500 ppm is equivalent to 63.86 and 74.26 mg/kg body weight/day for males and females, respectively
Critical effects observed:
no
Conclusions:
NOAEL (male/female): 1500 ppm (equivalent to 63.86 and 74.26 mg/kg body weight/day for males and females, respectively) (Based on adverse effects on body weight and nutritional parameters in male and female rats at 6000 ppm).
Executive summary:

The study was conducted according to guidelines, U.S. EPA, OPPTS 870.3100 and OECD 408 to evaluate the subchronic toxicity of the test article, when administered in the diet to rats for 13 weeks. Five groups of 10 animals/sex/group received untreated diet or the test article orally via dietary admixture at diet concentrations of 0, 100, 400, 1500, and 6000 ppm.

Observations for morbidity, mortality, injury, and the availability of food and water were conducted for all animals twice daily. Detailed clinical observations, including palpation for masses, were conducted predose on Day 1 and weekly thereafter during the study. Additional cageside observations were conducted once daily, except on the days of the weekly detailed clinical observation. Body weight was measured and recorded predose on Day 1 and weekly thereafter during the study. Food consumption was measured and recorded weekly. Body weight gain, food efficiency, and compound consumption were calculated. Ophthalmoscopic examinations were conducted pretest and prior to the terminal necropsy. Blood and urine samples for clinical pathology evaluations were collected and evaluated from all surviving animals prior to the terminal necropsy. Necropsy examinations were performed, organ weights were recorded, and designated tissues were collected for microscopic examination from all surviving animals at the terminal necropsy.

The overall (Days 1 – 91) mean compound consumption values of test substance in the 100, 400, 1500, and 6000 ppm groups were 4.17, 17.01, 63.86, and 257.09 mg/kg body weight/day, respectively, for male rats, and 5.13, 20.38, 74.26, and 278.14 mg/kg/day, respectively, for female rats.

No test article-related effects were noted in survival, clinical observations, masses, food efficiency, ophthalmoscopic findings, coagulation, clinical chemistry, urinalysis, macroscopic observations, or microscopic observations. One unscheduled euthanasia occurred in the 400 ppm (male) group on study Day 75. The cause of morbidity was peritoneal inflammation and was considered incidental.

Adverse, test article-related decreases (compared to control) in mean body weight parameters were observed in males and females at 6000 ppm. On study Day 91 mean body weight was decreased by 11.0% and 12.3% in males and females, respectively, at 6000 ppm compared with controls (both statistically significant). Weekly mean body weight changes were generally lower in both sexes at 6000 PPM compared to controls, with only occasional statistical significance. Overall (Days 1 – 91) mean body change at 6000 ppm was statistically significantly lower than controls (20% and 32% below controls in males and females, respectively).

Adverse, test article-related decreases (compared to controls) in food intake parameters were observed in both sexes at 6000 ppm, and correlated with the body weight effects. Overall (Days 1 – 91) mean food consumption of both sexes at 6000 ppm was statistically significantly lower (14.4% for males and 24.2% for females) compared to controls.

Non-adverse, test article-related effects included decreased food consumption in females at 1500 ppm (compared to controls), hematology effects (mild decreases in red cell mass and eosinophils in both sexes at 6000 ppm relative to controls) and organ weight changes (minimally increased liver weights in both sexes at 6000 ppm and increased uterus with cervix weights in females at 6000 ppm).

Therefore, under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for the study was 1500 ppm (63.86 and 74.26 mg/kg body weight/day for males and females, respectively). The NOAEL was based on adverse effects on body weight and nutritional parameters in male and female rats at 6000 ppm.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
other: U.S. EPA Health Effects Test Guidelines OPPTS 870.3050 (2000)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
no
Species:
mouse
Strain:
other: Crl:CD1(ICR)
Details on species / strain selection:
Mice have historically been used in safety evaluation studies for oral toxicity testing. The Crl:CD1(ICR) mouse was selected based on consistently acceptable health status and on extensive experience with this strain at test facility.
Sex:
male/female
Route of administration:
oral: feed
Details on route of administration:
The dietary route of administration was selected because it is a potential route of human exposure.
Vehicle:
other: LLC Certified Rodent LabDiet
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
33 and 34 days for males and females respectively
Frequency of treatment:
Daily
Dose / conc.:
200 ppm
Remarks:
Males: 34 mg/kg bw/d; Females: 41 mg/kg bw/d
Dose / conc.:
800 ppm
Remarks:
Males:129 mg/kg bw/d; Females: 161 mg/kg bw/d
Dose / conc.:
2 500 ppm
Remarks:
Males: 416 mg/kg bw/d; Females: 504 mg/kg bw/d
Dose / conc.:
7 000 ppm
Remarks:
Males: 1104 mg/kg bw/d; Females: 1343 mg/kg bw/d
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Decreases in red cell mass parameters observed in male and female mice fed dietary concentrations of 7000 ppm were likely test substance-related, but based on their minimal and transient nature, these changes were considered to be non-adverse.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Cholesterol (CHOL) was higher in male and female mice dosed with 7000 ppm (161% and 155% of control, respectively. These changes are statistically significant.
Test substance related, non-adverse effects on hepatic enzyme parameters were observed in male and female rats. Test substance increased hepatic peroxisomal β-oxidation activity, a measure of potential peroxisome proliferation, in female mice at 7000 ppm. Total hepatic microsomal cytochrome P450 enzyme content and cytochrome P450 4A1/2/3 were increased at 2500 and 7000 ppm in male and females. Cytochrome P450 2B1/2 was increased at 7000 ppm in males. Cytochrome P450 1A2 was increased at 2500 and 7000 ppm in females. Cytochrome P450 2E1 was increased at 2500 and 7000 ppm in males and at 200, 800, 2500, and 7000 ppm in females. The increases in hepatic enzymes were consistent with the non-adverse, adaptive response of increased metabolism that resulted in increased liver weights and hepatocellular hypertrophy.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were no adverse organ weight effects in males or females at any concentration. Test substance-related organ weight effects included an increase in liver weights (males and females (≥2500 ppm)), an increase in spleen weights (males (≥2500 ppm) and females (7000 ppm)), and a decrease in accessory sex organ (ASO) weights (males (≥2500 ppm)).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related gross observations were limited to dark discoloration of the livers of four of ten males fed the 7000 ppm test diet.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related microscopic findings included minimal to moderate centrilobular hepatocellular hypertrophy in the livers of both sexes (males (≥2500 ppm) and females (7000 ppm)), and a minimal to mild increase in splenic extramedullary hematopoiesis in males (7000 ppm).
Key result
Dose descriptor:
NOAEL
Effect level:
7 000 ppm
Sex:
male/female
Basis for effect level:
other: Based on the absence of adverse changes in any of the parameters assessed.
Remarks on result:
other: 1104 mg/kg bw/day and 1343 mg/kg bw/day for males and females respectively
Critical effects observed:
no
Conclusions:
NOAEL (male/female): 7000 ppm (1104 mg/kg bw/day and 1343 mg/kg bw/day for males and females respectively) (Highest concentration tested) based on the absence of adverse changes in any of the parameters assessed.
Executive summary:

The study was conducted according to guidelines, U.S. EPA Health Effects Test Guidelines OPPTS 870.3050 and OECD Guideline 407 to assess the potential repeated-dose toxicity of test substance in mice. Five groups of young adult male and female mice (10/sex/group) were administered diets that contained 0, 200, 800, 2500, or 7000 ppm test substance for approximately 28 days (33 and 34 days for males and females, respectively). Test substance was shown to be homogeneous and at targeted concentrations. Body weights, food consumption, and detailed clinical observations were evaluated weekly and acute clinical observations were evaluated daily. Hepatic biochemical parameters and clinical and anatomical pathology endpoints were evaluated at the end of the exposure period.

Mean daily intake of test substance in the 200, 800, 2500 and 7000 ppm groups was 34, 129, 416, and 1104 mg/kg body weight (bw)/day in males and 41, 161, 504, and 1343 mg/kg bw/day in females, respectively.

There were no adverse changes in clinical pathology parameters at any of the dietary concentrations tested. Decrease in red cell mass parameters observed in male and female mice fed dietary concentrations of 7000 ppm were likely test substance-related, but based on their minimal and transient nature, these changes were considered to be non-adverse..

No test substance-related deaths, clinical signs, or effects on body weight or nutritional parameters were observed at any concentration.

There were no adverse changes in anatomic pathology parameters at any concentration. Liver effects included hepatocellular hypertrophy (≥2500 ppm (males) and 7000 ppm (females)), increased liver weights (≥2500 ppm (males and females)), and dark discoloration of the liver (7000 ppm (males)). All test substance-related effects on the liver were consistent with the pharmacological induction of hepatic enzymes and were interpreted to be non-adverse. Increased extramedullary hematopoiesis (7000 ppm (males)) and increased spleen weights (≥2500 ppm (males) and 7000 ppm (females)) were considered secondary to non-adverse hematology changes. Accessory sex organ weights were decreased in males fed 2500 and 7000 ppm of the test substance.

Test substance related, non-adverse effects on hepatic enzyme parameters were observed in male and female rats. Test substance increased hepatic peroxisomal β-oxidation activity, a measure of potential peroxisome proliferation, in female mice at 7000 ppm. Total hepatic microsomal cytochrome P450 enzyme content and cytochrome P450 4A1/2/3 were increased at 2500 and 7000 ppm in male and females. Cytochrome P450 2B1/2 was increased at 7000 ppm in males. Cytochrome P450 1A2 was increased at 2500 and 7000 ppm in females. Cytochrome P450 2E1 was increased at 2500 and 7000 ppm in males and at 200, 800, 2500, and 7000 ppm in females. The increases in hepatic enzymes were consistent with the non-adverse, adaptive response of increased metabolism that resulted in increased liver weights and hepatocellular hypertrophy.

Under the conditions of test, 28-day feeding study in mice, the no-observed-adverse-effect level (NOAEL) for male and female mice was 7000 ppm (1104 mg/kg bw/day and 1343 mg/kg bw/day, respectively), the highest dietary concentration tested, based on the absence of adverse changes in any of the parameters assessed.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
other: U.S. EPA Health Effects Test Guidelines OPPTS 870.3050 (2000)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
Rats have historically been used in safety evaluation studies for oral toxicity testing. The Crl:CD(SD) rat was selected based on consistently acceptable health status and on extensive experience with this strain at test facility.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Males from Charles River Laboratories, Inc., Raleigh, North Carolina and females from Charles River Laboratories, Inc, Kingston New York.
- Females: nulliparous and nonpregnant; yes
- Age at study initiation: approximately 50 days old
- Weight at study initiation: Males: 210.5 to 217.8 grams, Female: 171.0 to 174.8 grams
- Housing: Animals were housed 2 or 3 per cage in solid-bottom caging with bedding and enrichment. Each cage rack contained only animals of one sex.
- Diet: PMI® Nutrition International LLC Certified Rodent LabDiet® 5002 except when fasted.
- Water: ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: Certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional requirements and not to exceed stated maximum concentrations of key contaminants, including specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. Water samples are analyzed for total bacterial counts, and the presence of coliforms, lead, and other contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-26 C°
- Humidity: 30-70 %
- Photoperiod (hrs dark / hrs light): 12
Route of administration:
oral: feed
Details on route of administration:
The dietary route of administration was selected because it is a potential route of human exposure.
Vehicle:
other: LLC Certified Rodent LabDiet
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Diet samples containing test substance at the concentrations of 200, 800, and 10000/20000 ppm prepared and submitted for homogeneity/concentration verification. Diet samples containing test substance at the concentrations of 200 and 20000 ppm prepared were submitted for room temperature stability analysis. A 0 ppm control diet sample was included with each set of samples for analysis.
Stability samples were analyzed on the day of receipt and after required room temperature storage. Study samples were stored frozen or at room temperature within the established stability range until analysis and the remainders were kept frozen after analysis.
Diet samples were extracted by acetonitrile or further diluted with diluted control sample. The concentrations of test substance were determined by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection.
The analysis results showed that the test substance was homogeneously mixed, at the targeted concentrations, and stable when stored at room temperature for up to 22 days in the diet from 200 to 20000 ppm. The test substance was not detected in the control samples
Duration of treatment / exposure:
30 and 31 days for males and females respectively.
Frequency of treatment:
Daily
Dose / conc.:
200 ppm
Remarks:
(Males: 17 mg/kg bw/d; Females: 16 mg/kg bw/d)
Dose / conc.:
800 ppm
Remarks:
(Males: 65 mg/kg bw/d; Females: 64 mg/kg bw/d)
Dose / conc.:
4 000 ppm
Remarks:
(Males: 309 mg/kg bw/d; Females: 317 mg/kg bw/d)
Dose / conc.:
10 000 ppm
Remarks:
(Males: 653 mg/kg bw/d; Females: 627 mg/kg bw/d)
No. of animals per sex per dose:
5
Control animals:
yes, plain diet
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily to detect moribund or dead animals and abnormal behavior and/or appearance and daily to detect acute clinical signs of systemic toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly once

BODY WEIGHT: Yes
- Time schedule for examinations: weekly once

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on days 30 and 31 for males and females, respectively
- Animals fasted: Yes
- Parameters checked in table No.1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Test days 30 and 31 for males and females, respectively
- How many animals: 50
- Parameters checked in table No.2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Test days 30 and 31 for males and females, respectively
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table No.3 were examined.

OTHER:
Thyroid Hormone Evaluation: yes
- Time schedule for examinations: Test Day 23
Parent/Metabolite Analysis: Yes
- Time schedule for examinations: Test Day 23
Biochemical Evaluation: yes
- Time schedule for examinations: Test Days 30 and 31
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes (see table No.4)
Statistics:
- Significance was judged at p < 0.05. Separate analyses were performed on the data collected for each sex.

- For parameters like body weight, body weight gain, food consumption, food efficiency, clinical pathology, organ weight the perliminary test for analysis is Levene’s test for homogeneityand Shapiro-Wilk test for normality. If preliminary test is significant, method of of statistical analysis transforms of the data to achieve normality and variance homogeneity were used. The order of transforms attempted was log, square-root, and rank-order. If the log and square-root transforms failed, the rank-order was used. If preliminary test is not significant, the method of statistical analysis is One-way analysis of variance followed by Dunnett's test.

For parameters like cytochrome P450 (total and isozymes), ß-Oxidation, UDPGT, Hormone levels, the preliminary test is Levene’s test for homogeneity and Shapiro-Wilk test for normality. If preliminary test is significant, method of Statistical Analysis is Kruskal-Wallis test followed by Dunn's test. If preliminary test is not significant, the method of statistical analysis is One-way analysis of variance followed by Dunnett's test.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males administered 20000/10000 ppm of the test substance exhibited adverse, test substance-related decreases in body weight parameters compared to controls. Statistically significant decreases in mean body weights and body weight gains were noted at 20000/10000 ppm throughout the test period. Final (Day 28) mean body weights and overall (Day 0-28) body weight gains at 20000/10000 ppm were 27% and 57%, respectively, lower than control (both statistically significant).
Females administered 20000/10000 ppm of the test substance exhibited adverse, test substance-related decreases in body weight parameters compared to controls. Statistically significant decreases in mean body weights (all intervals) and body weight gains (except at Day 3-5, Day 7-10, Day 10-14, Day 7-14 and Day 14-21 intervals) were noted at 20000/10000 ppm. Final (Day 28) mean body weights and overall (Day 0-28) body weight gains at 20000/10000 ppm were 17% and 58%, respectively, lower than control (both statistically significant).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males administered 20000/10000 ppm of the test substance exhibited adverse, test substance-related decreases in nutritional parameters compared to controls. Statistically significant decreases in mean food consumption at 20000/10000 ppm compared to control were noted throughout the test period. Overall (Day 0-28) food consumption at 20000/10000 ppm was 36% lower (statistically significant) compared to control. At 4000 ppm, food consumption was significantly lower at all intervals except Day 7-14. These changes may be test substance-related, however, were not considered adverse.
Females administered 20000/10000 ppm of the test substance exhibited adverse, test substance-related decreases in nutritional parameters compared to controls. Statistically significant decreases in mean food consumption at 20000/10000 ppm compared to control were noted throughout the test period. Overall (Day 0-28) food consumption at 20000/10000 ppm were 36% lower (both statistically significant) compared to control.
The overall mean daily intake of test substance in the 200, 800, 4000, or 20000/10000 ppm groups was 17, 65, 309, and 653 mg/kg bw/day, respectively, for male rats and 16, 64, 317, and 627 mg/kg bw/day, respectively, for female rats.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Males administered 20000/10000 ppm of the test substance exhibited adverse, test substance-related decreases in nutritional parameters compared to controls. Overall (Day 0-28) food efficiency at 20000/10000 ppm was 33%, lower (both statistically significant) compared to control. At 4000 ppm, food efficiency was significantly lower only at Day 14-21 and 21-28. These changes may be test substance-related, however, were not considered adverse, as the decrease in overall (Day 0-28) food efficiency was not statistically significant compared to control.
Females administered 20000/10000 ppm of the test substance exhibited adverse, test substance-related decreases in nutritional parameters compared to controls. Overall (day 0-28) food efficiency at 20000/10000 ppm was 35%, lower (both statistically significant) compared to control.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Red cell mass parameters (red blood cell [RBC], hemoglobin [HGB] and hematocrit [HCT]) were minimally decreased in male rats fed 20000/10000 ppm (8-12%) and female rats fed 4000 or 20000/10000 ppm (8-11%) after 4 weeks of test substance exposure. The decreases in red cell mass were associated with an increase in red cell distribution width (RDW) in these same groups (7-10%). All these changes were statistically significant compared to controls. Based on the consistency of changes across red cell mass parameters, the red cell changes observed in males at 20000/10000 ppm and in females at ≥4000 ppm in the current 28-day study were considered to be related to exposure to the test substance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- Statistically significant changes in several clinical chemistry parameters, including glucose, cholesterol, blood urea nitrogen, and bilirubin, were observed in treated male and/or female groups and were considered test substance-related, but not adverse.
- Cholesterol (CHOL) was increased in male and female rats fed 20000/10000 ppm (135% and 176% of control, respectively; statistically significant). Although the individual values for CHOL were higher than the concurrent control, most were within the 95% historical control range (38-86 mg/dL in males, 48-101 mg/dL in females), and there were no statistically significant changes in serum triglycerides. Small changes in serum cholesterol are frequent findings in toxicology studies in rats and are generally believed to represent minor effects on lipid metabolism that do not adversely affect the health of the animals. The increase in cholesterol in the 20000/10000 ppm male and female groups is likely test substance-related but non- adverse.
- Bilirubin (BILI) was minimally lower in female rats fed 4000 or 20000/10000 ppm (75% and 66% of control, respectively; statistically significant). Since increases, rather than decreases, in bilirubin are associated with biologically significant effects, these decreases in BILI were not considered to be adverse. The decreases in BILI in the 4000 or 20000/10000 ppm female groups were likely secondary to induction of liver enzymes as suggested by increased liver weights and/or microscopic hepatocellular hypertrophy. Enzyme induction may enhance the metabolism and excretion of bilirubin, and total and individual cytochrome P450 enzymes were induced in the present study at ≥4000 ppm.
- Blood urea nitrogen (BUN) was minimally higher in male rats fed 20000/10000 ppm (152% of control). There were no correlative changes in serum creatinine or urinalysis parameters, nor in test substance-related microscopic changes in the kidneys in this group. In addition, no statistically significant changes in BUN occurred in female rats at any of the dietary concentrations tested. Therefore, the higher BUN in the 20000/10000 ppm male group was likely test substance related but was considered to be of uncertain relation to treatment but non-adverse.
- Glucose (GLUC) was elevated (statistically significant) in female rats fed ≥800 ppm. Although a test substance-related effect cannot be ruled out. In addition, in the current study, there were no statistically significant or dose-related changes in GLUC in male rats at any of the dietary concentrations tested. Therefore, these statistical differences in glucose in the treated female groups were most likely spurious and based on their minimal nature, are considered non-adverse.
- Under the conditions of this study, test substance also caused an increase in hepatic peroxisomal β-oxidation activity at 4000 and 20000/10000 ppm in male and female rats. Hepatic microsomal UDPGT activity and total cytochrome P450 enzyme content were increased at 20000/10000 ppm in males and at 4000 and 20,000/10,000 ppm in females. Cytochrome P450 isozymes 1A2 and 2B1/2 were increased at 20000/10000 ppm in males and females.
- The effects on hepatic enzymes at 4000 and 20000/10000 ppm were accompanied by statistically significant increases in relative (to final body weight) liver weight and liver hypertrophy. In the absence of clinical chemistry changes or anatomic pathology evidence of hepatic cellular injury, the changes in biochemical parameters were considered test substance-related but not adverse, and were consistent with an adaptive response of increased metabolism due to exposure to xenobiotics.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related increases in liver weights were observed in male and female rats fed diets containing 4000 and 20000/10000 ppm of the test substance. Since this increase in liver weights correlated with hepatocellular hypertrophy consistent with the induction of metabolic enzymes, it was considered non-adverse.
- Test substance-related decreases in male accessory sex organ (ASO) weights were also observed in rats fed diets containing 4000 and 20000/10000 ppm of the test substance. ASO weight effects were due to decreased fluid content in the seminal vesicles and coagulating glands and were interpreted to be secondary to decreased final body weights and not adverse.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related gross observations were recorded at necropsy in one male and three females fed the 20000/10000 ppm test diet. The male was observed to have small seminal vesicles, while the females had pale discoloration of the liver.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related microscopic findings included minimal to mild centrilobular hepatocellular hypertrophy in the livers of both sexes at 4000 ppm and above and decreased fluid in the seminal vesicles and coagulating glands of males at 20000/10000 ppm.
Other effects:
no effects observed
Description (incidence and severity):
The test substance did not induce changes in thyroid parameters that were consistent with the potential to modulate thyroid hormone homeostasis, when tested at dietary concentrations up to 20000/10000 ppm.
Key result
Dose descriptor:
NOAEL
Effect level:
309 mg/kg bw/day (nominal)
Based on:
other: Based on the effects on body weight, nutritional, and red cell mass parameters at 20000/10000 ppm.
Sex:
male
Key result
Dose descriptor:
NOAEL
Effect level:
64 mg/kg bw/day (nominal)
Based on:
other: Based on the effects on red cell mass parameters at 4000 ppm.
Sex:
female
Key result
Critical effects observed:
no
Conclusions:
NOAEL (Male): 4000 ppm (309 mg/kg bw/d) (Based on the effects on body weight, nutritional, and red cell mass parameters at 20000/10000 ppm).
NOAEL (Female): 800 ppm (64 mg/kg bw/d) (Based on the effects on red cell mass parameters at 4000 ppm).
Executive summary:

The study was conducted according to guidelines, U.S. EPA OPPTS 870.3050 and OECD Guideline 407 to assess the potential repeated-dose toxicity of test substance in rats. Five groups of young adult male and female rats, (5/sex/group) were administered diets that contained 0, 200, 800, 4000, or 20000 ppm test substance for approximately 28 days (30 and 31 days for males and females, respectively). The high concentration was reduced to 10000 ppm starting on test day 3, due to marked body weight loss in males and females at 20000 ppm. The test substance was shown to be homogeneous, at targeted concentrations, and stable in the diet. Body weights, food consumption, and detailed clinical observations were evaluated weekly and acute clinical observations were evaluated daily. Blood was collected during week 3 of test substance exposure for evaluation of serum thyroid hormone concentrations and for possible analysis of concentration of the test substance and metabolites in plasma. Hepatic biochemical parameters and clinical and anatomical pathology endpoints were evaluated at the end of the exposure period.

The overall mean daily intake of test substance in the 200, 800, 4000, or 20000/10000 ppm groups was 17, 65, 309, and 653 mg/kg body weight (bw)/day, respectively, for male rats and 16, 64, 317, and 627 mg/kg bw/day, respectively, for female rats.

No test substance-related deaths or clinical signs were noted.

Test substance-related, adverse effects were noted in body weight and nutritional parameters in males and females at 20000/10000 ppm. Statistically significant decreases in final (day 28) mean body weights and overall (day 0-28) body weight gains, food consumption, and food efficiency compared to controls were noted. At 4000 ppm, the body weight and nutritional parameters in males were generally lower but not considered adverse because of inconsistent dose response. No test substance-related changes were noted in the 200 or 800 ppm groups.

Decreases in red cell mass parameters (red blood cell, hemoglobin, and hematocrit) were present in male and female rats fed dietary concentrations of 20000/10000 ppm and in females fed 4000 ppm. These red cell changes were considered marginally adverse under the conditions of this study.

Test substance-related effects on accessory sex organs (ASO) were noted in males at ≥4000 ppm. The effects included decreased fluid in the seminal vesicles and coagulating glands (20000/10000 ppm), decreased accessory sex organ weights (≥4000 ppm) and small seminal vesicles (20000/10000 ppm). Due to the lack of a microscopic effect on the ASO tissues (except for fluid content), and the lack of an any ASO organ weight or microscopic finding in the 90-day feeding study in rats (at concentrations up to 6000 ppm), the decreased ASO fluid was not considered to be adverse. In addition, test substance-related increases in liver weights (males and females at ≥4000 ppm), pale discoloration of the liver (females at 20000/10000 ppm) and hepatocellular hypertrophy (males and females at ≥4000 ppm) were noted in the study. These changes were attributed to the induction of hepatic metabolic enzymes and were therefore interpreted to be not adverse.

No test substance-induced alterations were observed in T3, T4, or TSH concentrations.

Test substance related and non-adverse effects on hepatic enzyme parameters were observed in male and female rats at dietary concentrations of 4000 and 20000/10000 ppm. Hepatic peroxisomal β-oxidation activity, a measure of potential peroxisome proliferation, was increased in male and female rats at dietary concentrations ≥4000 ppm. Total hepatic microsomal cytochrome P450 enzyme content and UDPGT activity were increased in males at 20000/10000 ppm and in females at ≥4000 ppm. Cytochrome P450 1A2 and 2B1/2 were increased in males and females at 20000/10000 ppm. The increases in β-oxidation activity, UDPGT activity, and cytochrome P450 enzymes are consistent with the non-adverse, adaptive response of increased metabolism that resulted in increased liver weights and hepatocellulcar hypertrophy.

Under the conditions of the study, the no-observed-adverse-effect level (NOAEL) for test substance in males was 4000 ppm (309 mg/kg body weight/day) based on the effects on body weight, nutritional, and red cell mass parameters at 20000/10000 ppm. The NOAEL for females was 800 ppm (64 mg/kg body weight/day) based on the effects on red cell mass parameters at 4000 ppm.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity in Non-Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3150 (90-Day Oral Toxicity in Non-rodents)
Deviations:
no
Qualifier:
according to
Guideline:
other: MAFF Japan, 2-1-9 Notification 12 Nousan 8147, Agricultural Chemicals Regulation Laws (2000)
Deviations:
no
GLP compliance:
yes
Species:
dog
Strain:
Beagle
Details on species / strain selection:
The beagle was selected for use in this study because the beagle is the usual non-rodent model used for evaluating the toxicity of various test articles and for which there is a large historical database.
Sex:
male/female
Route of administration:
oral: feed
Details on route of administration:
The oral route is one of the potential routes of human exposure to this test article.
Vehicle:
other: ground meal Lab Diet
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Dose / conc.:
100 ppm
Remarks:
Male: 3.05 mg/kg bw/d; Female: 2.69 mg/kg bw/d
Dose / conc.:
400 ppm
Remarks:
Male: 12.20 mg/kg bw/d; Female: 12.15 mg/kg bw/d
Dose / conc.:
1 000 ppm
Remarks:
Male: 26.60 mg/kg bw/d; Female: 26.87 mg/kg bw/d
Dose / conc.:
4 000 ppm
Remarks:
Male: 114.94 mg/kg bw/d; Female: 131.13 mg/kg bw/d
No. of animals per sex per dose:
4
Control animals:
yes, plain diet
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related clinical observations of thinness (in two males and three females) were noted in the 4000 ppm group. The overall incidence of thinness in the 4000 ppm females was statistically significant (p <0.05). Thinness was also noted in one male at 100 ppm and two males at 400 ppm, but not in any 1000 ppm males or in females at 100, 400, or 1000 ppm.
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related effects on body weight parameters were noted in males at 4000 ppm and in females at ≥1000 ppm, when compared to controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Correlative test substance related effects on food consumption were primarily noted in males at 4000 ppm.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Correlative test substance-related effects on food efficiency were primarily noted in males at 4000 ppm and in females at ≥1000 ppm.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- At Weeks 8 and/or 13 in both sexes receiving 4000 ppm there were mild decreases in albumin (up to 27%) that also resulted in mild secondary decreases in calcium in males (up to 6%) relative to controls. Females at this dose also had mild increases in globulin (up to 43%), which when coupled with the albumin findings, is typical of an inflammatory pattern.
- At Week 13 in both sexes receiving 4000 ppm there were minimal increases in alkaline phosphatase (up to 116%) that were considered test article-related but may not be biologically relevant based on their small magnitude and lack of findings among other hepatobiliary markers. Also at Week 13 in males receiving 4000 ppm there were also mild decreases in creatinine (up to 24%) that were considered test article-related and secondary to reduced food consumption
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Organ weight changes occurring secondary to the marked test article-related body weight decrements and/or stress included the following: increased absolute and relative adrenal gland weights of males and females at 4000 ppm and decreased absolute and relative thymus weights of males and females at 4000 ppm; decreased absolute epididymides, absolute testes, and absolute and relative prostate weights in males at 4000 ppm, and decreased absolute and relative ovary and uterus with cervix weights in females at 4000 ppm. In addition, increased absolute and relative spleen weights in females at 4000 ppm were considered secondary to the hematological effects observed in this group.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Secondary stress-related test article-related macroscopic findings were limited to mild small thymus size in males at ≥1000 ppm. Small thymus size correlated with microscopic generalized lymphoid depletion in both males and females at ≥1000 ppm. Moderate body fat depletion was observed in 1 of 4 males and females (unscheduled death animal) at 4000 ppm. The body fat depletion was considered likely related to test article administration and correlated with the body weight reductions observed predominantly in males at 4000 ppm and females at ≥1000 ppm and also correlated with microscopic findings observed in males and females at 4000 ppm.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic findings considered secondary to the marked test article-related decrements in body weight, decreased food intake, and systemic stress were observed in the adrenal glands (diffuse cortical hypertrophy) of males and females at 4000 ppm; lymphoid system (generalized lymphoid depletion of the spleen, mandibular and/or mesenteric lymph nodes) of males and females at 4000 ppm; increased severity (but not incidence) of generalized lymphoid depletion of the thymus of males and females at >1000 ppm; and immaturity of the reproductive tract of males and females at 4000 ppm (testes, epididymides, prostate, ovary, and uterus with cervix). Microscopic findings considered secondary and/or responsive to the reported decreased red cell mass/hemolysis, and non-adverse included: erythrocytic hyperplasia of the bone marrow (femur, rib and/or sternum) of males at ≥1000 ppm and females at 4000 ppm; liver (increased Kupffer cell pigment and/or extramedullary hematopoiesis (EMH)) of males and/or females at 4000 ppm;
and increased incidence and/or severity of pigmented macrophage presence and/or EMH in the spleen of males and/or females at 4000 ppm.
Other effects:
no effects observed
Description (incidence and severity):
- There were no test substance-related neurobehavioral observations.
Key result
Dose descriptor:
NOAEL
Effect level:
400 ppm
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
food efficiency
organ weights and organ / body weight ratios
Remarks on result:
other: This NOAEL is equivalent to 12.20 and 12.15 mg/kg bw/day in males and females, respectively.
Critical effects observed:
no
Conclusions:
NOAEL (mlaes/females) is 400 ppm which is based on reduced thymic weights with increased severity of thymic lymphoid depletion at ≥1000 ppm in both males and females. In females, the NOAEL is also based on reductions in body weight and nutritional parameters at ≥1000 ppm.
Executive summary:

The study was conducted according to guidelines, U.S. EPA OPPTS 870.3150 and OECD 409 to evaluate the toxicity of test substance when administered via the diet for 13 consecutive weeks (90 consecutive days). Four treatment groups of four male and four female beagle dogs were administered the test article at respective dietary concentrations of 100, 400, 1000, and 4000 ppm. One additional group of four animals/sex served as the control and received the untreated canine diet. The treated or untreated canine diet was available to all groups once daily (for approximately 2 hours in the morning) for 90 consecutive days.

Observations for morbidity, mortality, injury, and the availability of food and water were conducted twice daily for all animals. Clinical observations were conducted daily beginning on Day -2. Neurobehavioral findings were conducted pretest then weekly. Body weights were measured and recorded twice pretest and then weekly. Food consumption was measured and recorded daily, and food efficiency was calculated. Compound consumption was calculated weekly. Ophthalmoscopic examinations were conducted on all animals pretest and all surviving animals prior to the terminal necropsy. Physical examinations were conducted on all animals pretest and all surviving animals prior to the terminal necropsy. Blood and urine samples for clinical pathology evaluations were collected from all animals pretest and all surviving animals during Weeks 4, 8, and 13 (prior to the terminal necropsy on Day 91). Blood samples for determination of the plasma concentrations of the test article were collected from all surviving animals during Week 9. At study termination, necropsy examinations were performed, organ weights were recorded, and tissues were microscopically examined.

The overall mean daily intake values of test substance for male dogs in the 100, 400, 1000, and 4000 ppm groups were 3.05, 12.20, 26.60, and 114.94 mg/kg body weight (bw)/day, respectively. The mean daily intake values for females in these dose groups were 2.69, 12.15, 26.87, and 131.13 mg/kg bw/day, respectively.

There were no test article-related effects on ophthalmosopic examinations.

All animals survived until their scheduled termination, with one exception. A single female at 4000 ppm was euthanized in extremis on Day 77. The cause of death was considered related to hemorrhage of undetermined origin; the relationship between the test substance and the hemorrhage/moribundity was uncertain, as hemorrhage was not observed in any other animals.

There were no test substance-related neurobehavioral observations or trends noted during the study. Test substance-related increases in clinical observations of thinness were noted in multiple dogs in the 4000 ppm group, and were correlated with similar findings noted at terminal physical examination. Numerous dogs in other groups (including control) were noted to be thin but the level of severity was less than that observed in 4000 ppm dogs. Other observed clinical signs were seen in other dose groups and/or did not display a consistent dose-response, and were of uncertain relationship to test article exposure.

Test substance-related effects on body weight parameters were noted in males at 4000 ppm and in females at ≥1000 ppm, when compared to controls. Although body weights males in other treatment groups were lower than in control, they did not display a dose response over a 10 fold increase in concentrations and similar effects were not noted in females at these concentrations. Therefore, their relationship to test article exposure was uncertain and they were not considered adverse. Correlative test substance-related effects on nutritional parameters (food consumption and/or food efficiency) were primarily noted in males at 4000 ppm and in females (food efficiency only) at ≥1000 ppm, and generally followed effects on body weight gain.

There were no test substance-related effects on coagulation or urinalysis parameters. At 4000 ppm, there were mild, adverse decreases in red cell mass that may have been due to a hemolytic process, as supported by alterations to erythrocyte morphology and associated microscopic findings in the bone marrow, spleen, and liver. There was also evidence of a mild inflammatory pattern among serum proteins (mild decreases in albumin and calcium, and increases in globulin) at this dose level, although these findings lacked definitive histologic correlates.

Effects noted on body weight and nutritional parameters were associated with expected secondary changes in organ weights and with macroscopic and microscopic changes. Secondary stress-related test article-related macroscopic findings were limited to mild small thymus size in one male/group at ≥1000 ppm. Small thymus size correlated with increased severity of microscopic generalized lymphoid depletion in both sexes at ≥1000 ppm. Moderate body fat depletion was observed in a few dogs at 4000 ppm and was considered likely related to test article administration in these animals, as it correlated with the body weight reductions and microscopic findings.

Organ weight changes occurring secondary to the marked test article-related body weight decrements and/or stress were observed in numerous organs in animals at 4000 ppm, including adrenal gland, thymus, epididymides, testes, prostate, and ovary and uterus with cervix. Increased absolute and relative spleen weights in females at 4000 ppm were considered secondary to hematological effects observed in this group.

No primary test article-related adverse microscopic pathology findings were observed. However, microscopic findings considered secondary to the marked test article-related decrements in body weight, decreased food intake, and systemic stress were observed in some organs, including adrenal glands (diffuse cortical hypertrophy of both sexes at 4000 ppm); lymphoid system (generalized lymphoid depletion of the spleen, thymus, mandibular and/or mesenteric lymph nodes of both sexes at 4000 ppm); increased severity (but not incidence) of generalized lymphoid depletion of the thymus of males and females at >1000 ppm; and immaturity of the reproductive tract of males and females at 4000 ppm (testes, epididymides, prostate, ovary, and uterus with cervix). Microscopic findings considered secondary and/or responsive to the reported decreased red cell mass/hemolysis, and not adverse, included: erythrocytic hyperplasia of the bone marrow (femur, rib and/or sternum) of males at ≥1000 ppm and females at 4000 ppm; liver (increased Kupffer cell pigment and/or extramedullary hematopoiesis (EMH)) of males and/or females at 4000 ppm; and increased incidence and/or severity of pigmented macrophage presence and/or EMH in the spleen of males and/or females at 4000 ppm.

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for test substance was 400 ppm in male and female dogs. In both males and females, the NOAEL is based on reduced thymic weights with increased severity of thymic lymphoid depletion at 1000 ppm. In females, the NOAEL is also based on reductions in body weight and nutritional parameters at 1000 ppm. This NOAEL is equivalent to 12.20 and 12.15 mg/kg bw/day in males and females, respectively. The effects at 4000 ppm are considered to have exceeded a maximum tolerated dose for both sexes.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
The test substance is administered orally through feed daily to several groups of experimental animals, one dose level per group for a period of 28 days. During the period of administration, the animals are observed closely, each day for signs of toxicity. Animals that die or are euthanized during the test are necropsied and at the conclusion of the test surviving animals are euthanized and necropsied. The data derived from the study should allow for the characterization of the test substance toxicity, for an indication of the dose response relationship and the determination of the No-Observed Adverse Effect Level (NOAEL).
GLP compliance:
no
Species:
dog
Strain:
Beagle
Details on species / strain selection:
Beagle is selected because it is the usual non-rodent model used for evaluating the toxicity of various test articles and for which there is a large historical database.
Sex:
male/female
Route of administration:
oral: feed
Details on route of administration:
The oral route is one of the potential routes of human exposure to this test substance.
Vehicle:
other: canine diet
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Dose / conc.:
300 ppm
Remarks:
Males: 6.08 mg/kg bw/d; Females: 8.18 mg/kg bw/d
Dose / conc.:
3 000 ppm
Remarks:
Males: 49.52 mg/kg bw/d; Females: 67.07 mg/kg bw/d
Dose / conc.:
10 000 ppm
Remarks:
Males: 55.10 mg/kg bw/d; Females: 145.04 mg/kg bw/d
For the high dose group (hereafter referred as 10000 ppm), the animals received 30000 ppm for Week 1, 15000 ppm for Week 2, and 10000 ppm for Weeks 3 and 4. The dietary concentration was decreased to due to very low food and severe body weight loss in this group.
No. of animals per sex per dose:
2
Control animals:
yes, plain diet
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test article-related clinical observations were primarily noted in the 10000 ppm male and/or female groups, and included decreased activity, thin appearance, inappetence, effects on feces, loss of skin elasticity, eyelids partially/completely closed, and cold skin.. Isolated incidences of vomitus were also noted in males in this group. These signs were generally considered secondary to the reduced food consumption and body weights. Clinical findings noted in other dose groups were generally negligible in comparison to the high-dose group. At the terminal veterinary physical examinations, all three surviving animals at 10000 ppm and 3 of 4 control animals were noted to be thin. This finding was also noted during weekly clinical observations in dogs in all groups except the 300 ppm group.
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related effects on body weight parameters were noted in both sexes at 10000 ppm and, to a lesser extent at 3000 ppm, when compared to controls. Final body weight in male and female 10000 ppm groups and in the 3000 ppm male group were lower than control. Final body weight in 3000 ppm females was comparable to control. The total (Day 1-29) mean body weight loss was higher in males and females at 3000 ppm and 10000 ppm compared to same sex controls, and exhibited a dose dependent pattern of effect and magnitude. The body weight effects were considered adverse at 10000 and 3000 ppm. Animals in all groups (including controls) lost weight over multiple weekly intervals (including pretest), and all groups except 300 ppm males and females had overall (Day 1-29) body weight loss. Dogs in the 10000 ppm and 3000 ppm groups lost more weight than controls over most weekly intervals.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Test substance-related effects on nutritional parameters were primarily noted in both sexes at 10000 ppm, and to a much lesser extent at 3000 ppm. The magnitude of decrease in food consumption for the overall Day 1 to 29 interval was 77.3% and 63.7% for males and females at 10000 ppm, respectively, and 32.3% and 11.1% for males and females at 3000 ppm, respectively, when compared to concurrent controls. Overall Day 1 to 29 mean food consumption for 300 ppm was slightly lower (10.2%) for males, but slightly higher (14.3%) for females when compared to concurrent controls. The effects noted at 3000 and 10000 ppm were considered adverse.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related effects on nutritional parameters were primarily noted in both sexes at 10000 ppm, and to a much lesser extent at 3000 ppm. Total feed efficiency was higher than concurrent controls in treated animals of both sexesadministered 300 ppm, but was lower than concurrent controls in both sexes at 3000 ppm and 10000 ppm.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Mlaes and females receiving 10000 ppm there was evidence of mild hepatic effects and/or dysfunction as illustrated by mild increases in ALT and bile acids (females only) relative to controls. The females at this dose also exhibited mild decreases in total protein and albumin.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Gross pathological findings:
effects observed, non-treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Indirect test article-related microscopic findings likely associated with body weight loss and/or nonspecific toxicity (stress) were present in testes/epididymides, skeletal muscle, salivary glands, thymus, kidneys (females only), and liver (females only) and included in the following:
- One male at 10000 ppm had minimal bilateral degeneration of spermatocytes/spermatids in seminiferous tubules of the testes and bilateral oligospermia/germ cell debris in the epididymides.
- Atrophy of the myofibers (biceps femoris muscle) and salivary glands (mandibular, parotid, and sublingual) was present in the male and females at 10000 ppm.
- One male at 10000 ppm had unilateral dilation/inflammation of the sublingual gland duct.
- An increased severity of decreased lymphocytes in the thymus was present in the male and females at 10000 ppm.
- Females at 10000 ppm had fatty change in tubules of the kidneys and fatty change in the liver both of which were likely associated with body weight loss and altered fat mobilization.
- One female at 300 ppm had chronic–active inflammation in the lung.
Key result
Dose descriptor:
NOAEL
Effect level:
300 ppm
Based on:
other: Based on effects on body weight and nutritional parameters at 3000 ppm and above.
Remarks on result:
other: Equivalent to 6.08 and 8.18 mg/kg/day in males and females respectively
Critical effects observed:
no
Conclusions:
NOAEL (males/ females) is 300 ppm (equivalent to 6.08 and 8.18 mg/kg/day in males and females, respectively) is based on body weight and nutritional parameters at 3000 ppm and above.
Executive summary:

The Study was conducted to evaluate the toxicity and any potential palatability issues of test substance when administered via the diet for 28 consecutive days. Three treatment groups of two male and two female beagle dogs were administered the test substance at respective dietary concentrations of 300, 3000, and 30000/15000/10000 ppm. One additional group of two dogs/sex served as the control group and received untreated canine diet. For the high dose group (hereafter referred to in the text as 10000 ppm), the animals received 30000 ppm for Week 1, 15000 ppm for Week 2, and 10000 ppm for Weeks 3 and 4. The dietary concentration was decreased to due to very low food consumption and severe body weight loss in this group. The treated or untreated canine diet was available to all groups once daily (for approximately 2 hours in the morning) for 28 consecutive days.

Observations for morbidity, mortality, injury, and the availability of food and water were conducted twice daily for all animals. Clinical observations were conducted daily beginning on Day-7. Neurobehavioral findings were conducted pretest then weekly. Body weights were measured and recorded twice pretest then weekly. Food consumption was measured and recorded daily, and food efficiency was calculated. Compound consumption was calculated weekly. Ophthalmoscopic and physical examinations were conducted on all animals pretest and all surviving animals prior to the terminal necropsy. Blood and urine samples for clinical pathology evaluations were collected from all animals pretest and all surviving animals prior to the terminal necropsy. At study termination, necropsy examinations were performed and organ weights were recorded. Tissues were microscopically examined for animals at 0, 3000, and 10000 ppm.

The overall mean daily intake values of test substance for male dogs in the 300, 3000, and 10000 ppm groups were 6.08, 49.52, and 55.10 mg/kg/day, respectively. The mean daily intake values for females in these dose groups were 8.18, 67.07, and 145.04 mg/kg/day, respectively.

There were no test article-related effects on ophthalmoscopic examinations.

All animals survived until their scheduled termination, with one exception. A single male from the 10000 ppm group (animal number 815) was euthanized in extremis on Day 24 based on severe inappetence, body weight loss, and clinical signs, which progressed over time. Based on clinical pathology and microscopic pathology findings, this animal was determined to have acute bacterial pneumonia, considered unrelated to test article exposure.

Test article-related clinical observations were primarily noted in the 10000 ppm male and/or female groups, and included decreased activity, thin appearance, inappetence, effects on feces, loss of skin elasticity, eyelids partially/completely closed, and cold skin.. Isolated incidences of vomitus were also noted in males in this group. These signs were generally considered secondary to the reduced food consumption and body weights. Clinical findings noted in other dose groups were generally negligible in comparison to the high-dose group. At the terminal veterinary physical examinations, all three surviving animals at 10000 ppm and 3 of 4 control animals were noted to be thin. This finding was also noted during weekly clinical observations in dogs in all groups except the 300 ppm group.

There were no definitive test substance-related neurobehavioral observations or trends noted during the study in groups exposed to up to 3000 ppm. A few findings noted at 10000 ppm (including the male euthanized on day 24) were attributed to the body weight loss and decreased food consumption noted in this group.

Test substance-related effects on body weight parameters were noted in both sexes at 10000 ppm and, to a lesser extent at 3000 ppm, when compared to controls. Final body weight in male and female 10000 ppm groups and in the 3000 ppm male group were lower than control. Final body weight in 3000 ppm females was comparable to control. The total (Day 1-29) mean body weight loss was higher in males and females at 3000 ppm and 10000 ppm compared to same sex controls, and exhibited a dose dependent pattern of effect and magnitude. The body weight effects were considered adverse at 10000 and 3000 ppm. Animals in all groups (including controls) lost weight over multiple weekly intervals (including pretest), and all groups except 300 ppm males and females had overall (Day 1-29) body weight loss. In general, dogs in the 10000 ppm and 3000 ppm groups lost more weight than controls over most weekly intervals.

Test substance-related effects on nutritional parameters (food consumption and food efficiency) were noted in both sexes at 10000 ppm, and to a much lesser extent at 3000 ppm, and were considered adverse. Effects on food efficiency generally followed effects on body weight gain. The reduction in the high-dose group dietary concentration was generally associated with less severe effects on body weight and nutritional parameters, but these parameters remained below that of control, especially in males. Therefore, the 10000 ppm concentration was considered to have exceeded the maximum tolerated dose.

There were no test substance-related effects on hematology, coagulation, or urinalysis parameters or adverse effects on clinical chemistry parameters. There were minimal decreases in red cell mass in all groups (including controls) at the termination interval that were considered due to blood draws and not relevant to the test article. At termination in both sexes receiving 10000 ppm there was evidence of mild hepatic effects and or dysfunction as illustrated by mild decreases in total protein and albumin. These findings were considered test article-related but not adverse.

There were no test susbtance-related macroscopic observations or definitive direct test substance-related organ weight changes. There were no test article-related microscopic observations in animals at 3000 ppm or below. At 10000, indirect test article-related microscopic findings, likely associated with severe body weight loss and/or nonspecific toxicity (stress), were present in multiple organs, including testes/epididymides, skeletal muscle, salivary glands, thymus, kidneys (females only), and liver (females only) and were considered adverse. Fatty change in tubules of the kidneys and in the liver of females at 10000 ppm were likely associated with body weight loss and altered fat mobilization.

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for test substance in male and female dogs was 300 ppm, based on effects on body weight and nutritional parameters at 3000 ppm and above. This NOAEL is equivalent to 6.08 and 8.18 mg/kg/day in males and females, respectively. Reduction in the high-dose group dietary concentration was generally associated with less severe effects on body weight and nutritional parameters, but these parameters remained below that of control, especially in males. Therefore, this concentration was considered to have exceeded the maximum tolerated dose. Effects that were observed in the high dose group were generally attributed to severe systemic toxicity due to exposure to this excessive dose, and not to specific target organ toxicity. This conclusion is supported by the lack of similar findings at lower concentrations where body weight and nutritional effects of similar magnitude were not observed.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))
Deviations:
no
Qualifier:
according to
Guideline:
other: MAFF Japan Agricultural Chemicals Regulation Laws 2-1-10 Notification 12 Nousan 8147 (2000)
Deviations:
no
GLP compliance:
yes
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The Crl:CD(SD) rat was selected based on consistently acceptable health status and on extensive experience with the strain at test facility.
Sex:
male/female
Type of coverage:
semiocclusive
Vehicle:
water
Remarks:
Deionized
Duration of treatment / exposure:
29 days
Frequency of treatment:
Daily
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
350 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Clinical signs:
no effects observed
Dermal irritation:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Ophthalmological findings:
effects observed, non-treatment-related
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
other: Based on the lack of test substance-related effects on any in-life, clinical pathology, or anatomic pathology parameters in rats treated dermally up to 1000 mg/kg/day.
Sex:
male/female
Conclusions:
NOAEL (males/females): 1000 mg/kg bw/d (Highest concentration tested) (based on the lack of test substance-related effects on any in-life, clinical pathology, or anatomic pathology parameters)
Executive summary:

The study was conducted according to guidelines, U.S. EPA OPPTS 870.3200 and OECD Guideline 410 to assess the potential repeated-dose dermal toxicity of test substance in rats. Four groups of young adult male and female rats (10/sex/group) were administered the test substance dermally for approximately 6 hours/day at dosages of 0, 100, 350, or 1000 mg/kg body weight (bw)/day for 29 days. Body weights, food consumption, and detailed clinical observations were evaluated weekly. The animals were evaluated for acute signs of systemic toxicity on days detailed clinical observations were not conducted. Ophthalmological evaluations were performed prior to the start of dermal exposure and near the end of the exposure period. Clinical pathology endpoints (hematology, coagulation, clinical chemistry, and urinalysis) were evaluated at the end of the exposure period. On test Day 29, the rats were sacrificed and given a gross and microscopic pathological evaluation.

No deaths occurred and no clinical or ophthalmological observations were attributed to exposure of the test substance. No adverse or test substance-related effects on body weight or nutritional parameters were observed.

There were no test substance-related effects on clinical pathology parameters, organ weights, gross pathology, or microscopic pathology.

The no-observed-adverse-effect level (NOAEL) for male and female rats was 1000 mg/kg bw/day. The NOAEL was based on the lack of test substance-related effects on any in-life, clinical pathology, or anatomic pathology parameters in rats treated dermally up to 1000 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))
Deviations:
no
Qualifier:
according to
Guideline:
other: MAFF Japan Agricultural Chemicals Regulation Laws 2-1-10 Notification 12 Nousan 8147 (2000)
Deviations:
no
GLP compliance:
yes
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The Crl:CD(SD) rat was selected based on consistently acceptable health status and on extensive experience with the strain at test facility.
Sex:
male/female
Type of coverage:
semiocclusive
Vehicle:
water
Remarks:
Deionized
Duration of treatment / exposure:
29 days
Frequency of treatment:
Daily
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
350 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Clinical signs:
no effects observed
Dermal irritation:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Ophthalmological findings:
effects observed, non-treatment-related
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
other: Based on the lack of test substance-related effects on any in-life, clinical pathology, or anatomic pathology parameters in rats treated dermally up to 1000 mg/kg/day.
Sex:
male/female
Conclusions:
NOAEL (males/females): 1000 mg/kg bw/d (Highest concentration tested) (based on the lack of test substance-related effects on any in-life, clinical pathology, or anatomic pathology parameters)
Executive summary:

The study was conducted according to guidelines, U.S. EPA OPPTS 870.3200 and OECD Guideline 410 to assess the potential repeated-dose dermal toxicity of test substance in rats. Four groups of young adult male and female rats (10/sex/group) were administered the test substance dermally for approximately 6 hours/day at dosages of 0, 100, 350, or 1000 mg/kg body weight (bw)/day for 29 days. Body weights, food consumption, and detailed clinical observations were evaluated weekly. The animals were evaluated for acute signs of systemic toxicity on days detailed clinical observations were not conducted. Ophthalmological evaluations were performed prior to the start of dermal exposure and near the end of the exposure period. Clinical pathology endpoints (hematology, coagulation, clinical chemistry, and urinalysis) were evaluated at the end of the exposure period. On test Day 29, the rats were sacrificed and given a gross and microscopic pathological evaluation.

No deaths occurred and no clinical or ophthalmological observations were attributed to exposure of the test substance. No adverse or test substance-related effects on body weight or nutritional parameters were observed.

There were no test substance-related effects on clinical pathology parameters, organ weights, gross pathology, or microscopic pathology.

The no-observed-adverse-effect level (NOAEL) for male and female rats was 1000 mg/kg bw/day. The NOAEL was based on the lack of test substance-related effects on any in-life, clinical pathology, or anatomic pathology parameters in rats treated dermally up to 1000 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Additional information

The primary toxicological findings in short-term, repeated-dose oral toxicity studies in rodents included changes in body weight and nutritional parameters, increases in liver weight, and hepatocellular hypertrophy. The liver weight effects in rats and mice occurred in the absence of clinical or histopathological findings indicative of liver toxicity. Increases in cytochrome P450 liver enzyme content (total and specific isozymes), beta-oxidation activity, and mild reductions in bilirubin (rats) were noted in 28-day and/or 90-day studies in rats and mice, suggesting a non-adverse, adaptive, pharmacological response to increased metabolism due to exposure to the test substance. Hepatocellular hypertrophy, characterized as increased amounts of finely granular eosinophilic cytoplasm within centrilobular hepatocytes, accompanied the increases in liver weights in rats and mice (28 days and 90 days). Male rats and mice were more sensitive to the liver effects than females. In rats, these liver effects were also associated with increased liver UDP-glucuronyltransferase activity (UDP-GT). However, the effects on UDP-GT were considered non-adverse as there were no correlative effects on thyroid hormones or thyroid pathology.

 

A few other effects were observed in the 28-day studies in rats and mice at higher concentrations, including reduced RBC mass parameters, and reduced accessory sex organ weight with reduced seminal vesicle and coagulating gland fluid. However, these effects were considered non-adverse as they were transient (differences were observed in the 28-day studies only, not in the longer term studies) and may have been secondary to marked body weight and nutritional effects observed at these concentrations.

 

A second 90-day rat feeding study was conducted with a test batch representing the proposed manufacturing process to bridge to the toxicology findings from studies conducted with the test substance manufactured by an earlier process. In the bridging study, test substance-related effects were generally similar to those observed in the original study. The NOAEL for both sexes was the same in both studies and was based on body weight and nutritional effects of similar magnitude. Non-adverse increases in liver weights were observed in both studies; however, a small increase in hepatocellular hypertrophy observed in males in the original study was not observed in the bridging study. Therefore, there were no notable differences in the subchronic toxicity profile between the materials produced by the two manufacturing processes.

 

Dogs were the most sensitive species to the test substance via repeated-dose toxicity. The primary effects observed in the 28-day, 90-day, and 1-year feeding studies were reductions in body weight and nutritional parameters. At the highest dietary concentrations (10000 ppm for 28 days and 4000 ppm for 90 days), effects supported that these concentrations exceeded the MTD. No specific target organ was identified; most clinical and anatomic pathology effects were considered secondary to marked body weight and nutritional effects and associated non- specific stress. In the 28-day and 90-day dog studies, clinical chemistry effects were suggestive of liver dysfunction, but no specific correlative liver histopathology was observed, and clinical and anatomic evidence of liver toxicity was not observed in the 90-day study. An increase in the severity (but not the incidence) of thymus lymphoid depletion, with associated reduction in thymus weight, was observed in dogs at ≥1000 ppm in the 90-day study, but these effects were not observed at 2000 ppm in the 1-year dog study. Effects in the 90-day and 1-year dog studies were not consistent as effects observed at 1000 ppm in the 90-day study were not observed at 2000 ppm in the 1-year study (thymus effects; body weight and nutritional effects). The reason for this inconsistency is not understood.

 

In a 90-day dog study, one 4000 ppm female was euthanised in extremis on Day 77, due to severe clinical signs attributed to haemorrhage of unknown origin. The relationship between this death and test substance exposure is uncertain. Vascular arteritis was observed in one female dog sacrificed prior to scheduled sacrifice, at 400 ppm in the 1-year study. This condition was attributed to spontaneous Canine Juvenile Polyarteritis and not to test substance exposure. The NOAEL in dogs is 400 ppm, equivalent to 12.20 and 12.15 mg/kg bw/day in males and females, respectively, based on the results in the 90-day study.

Dermal application of the test substance to rats for 28 days did not result in any treatment-related findings up to and including a maximum dose of 1000 mg/kg bw/day.

Justification for classification or non-classification

Under the criteria of CLP Regulation [EC] No. 1272/2008, STOT RE may be assigned on the basis of a substance demonstrating evidence of significant or severe specific organ toxicity in a 90-day oral study at or below a guidance value of 100 mg/kg bw/day (basis of Category 2). This guidance value is adjusted in accordance with the Haber’s rule for studies of different durations. ‘Significant’ toxicity is taken to mean changes that clearly indicate functional disturbance or morphological changes that are toxicologically relevant. ‘Severe’ toxicity is considered to be more profound or serious and indicates changes that are of a considerably adverse nature with a significant impact on health. There is no evidence for any adverse findings or serious target organ toxicity in 90-day repeat dosing studies in rats, mice or dogs and in a 1-year feeding study in dogs that meet the criteria of CLP Regulation [EC] No. 1272/2008 for STOT RE. The primary effects in these feeding studies were changes in body weight and nutritional parameters, increases in liver weight, and hepatocellular hypertrophy in mice and rats and were observed at high doses. In the absence of liver cell toxicity, these changes are attributed to no adverse induction of hepatic metabolising enzymes, an adaptive response to xenobiotic exposure. The test substance did not induce any adverse effects when tested by the dermal route in short-term repeat exposure studies. Dogs were the most sensitive species to the test substance via repeated dose toxicity. The primary effects observed in the dog studies were reductions in body weight and nutritional parameters. At high concentrations, the clinical and anatomic pathology effects observed were considered secondary to marked body weight and nutritional effects and associated with non-specific stress. No specific target organ toxicity was identified. In the 28-day and 90-day dog studies, clinical chemistry effects were suggestive of liver dysfunction, but no specific correlative liver histopathology was observed, and clinical and anatomic evidence of liver toxicity was not observed in the 90-day study. An increase in the severity (but not the incidence) of thymus lymphoid depletion, with associated reduction in thymus weight, was observed in dogs at ≥ 1000 ppm in the 90-day study, but these effects were not observed at 2000 ppm in the 1-year dog study. Effects in the 90-day and 1-year dog studies were not consistent as effects observed at 1000 ppm in the 90-day study were not observed at 2000 ppm in the 1-year study (thymus effects; body weight and nutritional effects). Therefore, the test substance is not classified for STOT RE according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.