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Carcinogenicity

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Description of key information

2-Year Rat Diet: Not Carcinogenic. OECD 453; Reliability = 1

18-Month Mouse Diet: Not Carcinogenic. OECD 451; Reliability = 1

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Referenceopen allclose all

Endpoint:
carcinogenicity: 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 451 (Carcinogenicity Studies)
Deviations:
yes
Qualifier:
according to
Guideline:
EU Method B.32 (Carcinogenicity Test)
Deviations:
yes
Qualifier:
according to
Guideline:
EPA OPPTS 870.4200 (Carcinogenicity)
Deviations:
yes
Qualifier:
according to
Guideline:
other: JMAFF Guidelines for Data Requirements for Supporting Registration of Pesticides, No. 12-Nousan-No. 8147 (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
Vehicle:
other: Meal Lab Diet
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
At least 78 weeks
Frequency of treatment:
The test article was available in the diet ad libitum.
Dose / conc.:
200 ppm
Remarks:
(Male: 20.08 mg/kg bw/d; Female: 21.82 mg/kg bw/d)
Dose / conc.:
800 ppm
Remarks:
(Male: 84.49 mg/kg bw/d; Female: 88.04 mg/kg bw/d)
Dose / conc.:
2 500 ppm
Remarks:
(Male: 248.26 mg/kg bw/d); Female: 283.27 mg/kg bw/d)
Dose / conc.:
7 000 ppm
Remarks:
(Male: 726.72 mg/kg bw/d; Female: 809.75 mg/kg bw/d)
No. of animals per sex per dose:
60
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, non-treatment-related
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:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related organ weight changes were present in the liver in males at 7000 ppm and in females at 2500 and 7000 ppm, and in the spleen in females at 7000 ppm.
- Liver weights (absolute and relative to body and brain) were statistically significantly higher in males at 7000 ppm and in females at 2500 and 7000 ppm. The higher weights were related to an increased incidence of hepatocellular adenomas in males. There were no microscopic correlates in females.
- Spleen weights (absolute and relative to body and brain) were statistically significantly higher in females at 7000 ppm by 95.57%, 98.22%, and 94.98%, respectively, compared to controls. The higher spleen weights correlated to an increased incidence/severity of extramedullary hematopoiesis.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related macroscopic changes were present in the liver in males at 7000 ppm, and in the spleen in females at 7000 ppm.
- Mass or nodule was noted in the liver of 27/60 males at 7000 ppm compared to 12/60 males from the control group. The masses and nodules generally correlated to hepatocellular adenomas.
- Enlarged spleen was present in 15/60 females at 7000 ppm compared to 5/60 females from the control group. The finding correlated to an increase in severity/incidence of extramedullary hematopoiesis.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related non-neoplastic changes consisted of centrilobular hypertrophy in the liver in males and in females at 2500 and 7000 ppm, ductal dilatation in the mammary glands in females at 7000 ppm, increased extramedullary hematopoiesis in the spleen in males at 7000 ppm and in females at ≥800 ppm and in the liver in females at 7000 ppm, and increased severity of mucosal hyperplasia in the duodenum in males at 7000 ppm.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Definitive test substance-related neoplastic findings consisted of hepatocellular adenomas in the liver in males at 7000 ppm.
- The hepatocellular adenomas were considered secondary to enzyme induction in the liver, as evidenced by an increased incidence of centrilobular hypertrophy consistent with increases in hepatic microsomal enzymes that were observed in short-term feeding studies with the test article.
Relevance of carcinogenic effects / potential:
Test substance is not oncogenic at dietary concentrations up to and including 2500 ppm for males and 7000 ppm for females. Test-substance related neoplastic findings were limited to males only at the highest dietary concentration administered in this study, which was 7000 ppm.
Key result
Dose descriptor:
NOAEL
Effect level:
2 500 ppm
Sex:
male
Basis for effect level:
gross pathology
histopathology: neoplastic
Remarks on result:
other:
Remarks:
2500 ppm is equivalent to 248.3 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Effect level:
7 000 ppm
Sex:
female
Remarks on result:
other:
Remarks:
7000 ppm is equivalent to 809.8 mg/kg bw/day
Conclusions:
NOAEL (Male): 2500 ppm (equivalent to 248.3 mg/kg bw/day) (Anatomic pathology findings)
NOAEL (Female): 7000 ppm (equivalent to 809.8 mg/kg bw/day) (Highest concentration tested)
Executive summary:

The Study was conducted according to guidelines OECD Guideline 451and US EPA 870.4200 to evaluate the potential oncogenicity of the test substance, when incorporated into nutritionally adequate diet and administered over the major portion of the life span of mice.

Five groups of 60 animals/sex/group received untreated diet or the test article orally via dietary admixture for at least 78 weeks at diet concentrations of 0, 200, 800, 2500, and 7000 ppm. Observations for morbidity, mortality, injury, and the availability of food and water were conducted for all animals twice daily through Week 56 and three times daily thereafter. Detailed clinical and mass observations were conducted weekly during the study. Body weight and food consumption were measured and recorded weekly for the first 13 weeks of the study, then every other week thereafter. Body weight gain, food efficiency, and compound consumption were calculated. Ophthalmoscopic examinations were conducted pretest and prior to the terminal necropsy. Blood samples for clinical pathology evaluation (blood smears for differential blood counts) were collected and evaluated from all animals euthanized in extremis. Differential blood counts were evaluated from control and high dose animals at the end of the study, and the results did not warrant the evaluation of other groups. Necropsy examinations were performed, organ weights were recorded, and designated tissues were collected for microscopic examination from all surviving animals at the terminal necropsy. An additional 20 animals/sex were co-housed with study animals, and serological health screens were conducted on these animals pretest and at 6, 12, and 18 months.

The analytical results showed that the test article was homogeneously mixed in the diet and present at the targeted concentrations. Test substance was not detected in control samples.

No test substance-related effects were noted on the following parameters: survival, clinical findings or masses, body weight and food intake parameters, or ophthalmoscopic evaluations.

The overall (Week 1 to 77) mean compound consumption of test substance in the 200, 800, 2500, and 7000 ppm groups was 20.08, 84.49, 248.26, and 726.72 mg/kg body weight (bw)/day, respectively, for male mice and 21.82, 88.04, 283.27, and 809.75 mg/kg bw/day, respectively, for female mice.

An increase in the number of animals with mass or nodule in the liver was noted in males at 7000 ppm and correlated with the presence of hepatocellular adenomas. Liver weights were statistically significantly higher in males at 7000 ppm and in females at ≥2500 ppm and were related to the increased incidence of hepatocellular adenomas in males and an increased incidence of hepatocellular hypertrophy in males at ≥200 ppm and in females at ≥2500 ppm. Enlarged spleen noted in females at 7000 ppm correlated with increased spleen weights at 7000 ppm. An increase in severity/incidence of extramedullary hematopoiesis was observed in the spleen of males at 7000 ppm and in females at ≥800 ppm, and in the liver of females at 7000 ppm. However, in the absence of correlative changes in the bone marrow or other changes indicative of adverse effects on red blood cells, the changes in spleen and liver were considered to be nonadverse. A higher incidence of ductal dilation of the mammary gland occurred in the 7000 ppm females and consisted of dilated ducts with secretory material. Mucosal hyperplasia of the duodenum of moderate grade was increased in incidence in males at 7000 ppm.

Definitive test substance-related neoplastic findings consisted of hepatocellular adenomas in males at 7000 ppm. The hepatocellular adenomas were considered secondary to enzyme induction in the liver, as evidenced by an increased incidence of centrilobular hypertrophy consistent with increases in hepatic microsomal enzymes that were observed in short-term feeding studies with the test article.

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for test substance when administered to mice for 18 months is 2500 ppm for males, equivalent to 248.3 mg/kg bw/day, and 7000 ppm for females, equivalent to 809.8 mg/kg bw/day. The NOAEL is based on anatomic pathology findings at 7000 ppm in males only. Test substance is not oncogenic at dietary concentrations up to and including 2500 ppm for males and 7000 ppm for females. Test-substance related neoplastic findings were limited to males only at the highest dietary concentration administered (7000 ppm).

Endpoint:
carcinogenicity: 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 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
yes
Qualifier:
according to
Guideline:
EPA OPPTS 870.4300 (Combined Chronic Toxicity / Carcinogenicity)
Deviations:
yes
Qualifier:
according to
Guideline:
EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)
Deviations:
yes
Qualifier:
according to
Guideline:
other: JMAFF Guidelines for Data Requirements for Supporting Registration of Pesticides, No. 12-Nousan-No. 8147 (2000)
Deviations:
yes
GLP compliance:
yes
Species:
rat
Strain:
other: CD® [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
Vehicle:
other: Ground meal Lab Diet®
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Atleast 104 weeks (Additional animals, designated for the 12-month interim necropsy, received the control or test diet for approximately 52 weeks)
Frequency of treatment:
The test article was available in the diet ad libitum.
Dose / conc.:
100 ppm
Remarks:
(Male: 3.03 mg/kg bw/d; Female: 3.23 mg/kg bw/d)
Dose / conc.:
500 ppm
Remarks:
(Male: 15.92 mg/kg bw/d; Female: 17.34 mg/kg bw/d)
Dose / conc.:
2 000 ppm
Remarks:
(Male: 70.55 mg/kg bw/d; Female: 73.80 mg/kg bw/d)
Dose / conc.:
8 000 ppm
Remarks:
(Male: 283.83 mg/kg bw/d; Female: 395.88 mg/kg bw/d)
No. of animals per sex per dose:
70 animals/sex/group (Additional animals (10 animals/sex/group), designated for the 12-month interim necropsy
Control animals:
yes, plain diet
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
A greater number of females in the 8000 ppm group were noted to be thin in appearance relative to controls, which correlated with the marked reductions in body weight that occurred in these animals.
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Adverse, test article-related lower body weight and nutritional parameters were noted during the study in males and females at ≥2000 ppm. Effects observed in females were more severe than those observed in males exposed to the same dietary concentrations and the degree of change in absolute body weight (23.0 and 45.5% in 2000 and 8000 ppm females, respectively) were considered to have greatly exceeded a maximum tolerated dose for systemic toxicity.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean food consumption in 8000 ppm males was 12.5% and 13.1% below control over one and two years, respectively (both statistically significant). At 2000 ppm, mean food consumption for males was 2.6% and 1.9% below control (not statistically significant). Only the effects at 8000 ppm were considered adverse. In females, mean food consumption at 8000 ppm was 12% and 11% below control over one and two years (both statistically significant). Mean food consumption at 2000 ppm was 8% and 6.7% below control over one and two years (both statistically significant). Food consumption effects at both concentrations in females were considered test article related and adverse.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
In 8000 ppm males, mean food efficiency over the first year and over the entire study was 15.7% and 13.4% below control, respectively. In 2000 ppm males, mean food efficiency over the first year and over the entire study was 8.3% and 9.4% below control, respectively. Only the one year interval difference was statistically significant at both concentrations. In 8000 ppm females, mean food efficiency over the first year and over the entire study was 39.5% and 52.7% below control, respectively. In 2000 ppm females, mean food efficiency over the first year and over the entire study was 22.5% and 21.2% below control, respectively.
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Test article-related effects among hematology parameters for interim (12-month) animals included mild, non-adverse decreases in red cell mass and eosinophils in both sexes at 8000 ppm, with marginal effects on these parameters in animals receiving 2000 ppm. These findings lacked microscopic correlates and were not associated with increases in mean cell volume or reticulocyte count.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Test article-related effects among clinical chemistry parameters for interim (12-month) animals included mild increases in urea nitrogen and phosphorus in both sexes at 8000 ppm, mild decreases in chloride in both sexes at 8000 ppm, mild increases in cholesterol in females at 2000 and 8000 ppm, and increases in bile acids in females at 8000 ppm. These findings were minor and non-adverse; they had no microscopic correlates.
Urinalysis findings:
effects observed, non-treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At this interim necropsy, test article-related increased mean liver weights occurred in both males and females at 2000 and 8000 ppm. At the terminal necropsy, liver weights were only statistically significantly increased in females at 2000 and 8000 ppm. The increased liver weights correlated to hepatocellular hypertrophy noted in both sexes at 8000 ppm at the interim and terminal necropsies and in females at 2000 ppm at the terminal necropsy.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Following the terminal necropsy, test article-related macroscopic observations were noted in the lung of male and female rats and the uterus of females. In the lung there was an increased incidence of focus/foci at 8000 ppm in both sexes and at 2000 ppm only in females, which correlated microscopically with alveolar histiocytosis. In the uterus of females there was an increased incidence of enlarged uterus at 2000 and 8000 ppm and mean uterus weights were increased at 8000 ppm. Enlarged uterus was correlative to the presence of tumors at 8000 ppm and dilatation of the uterus with or without additional findings of endometrial hyperplasia, inflammation, or hemorrhage.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test article-related non-neoplastic microscopic findings at terminal necropsy occurred in the liver, lung, and testes of males at 8000 ppm and in females in the liver at 2000 and 8000 ppm, and the lung and uterus at 8000 ppm. In the liver of males at 8000 ppm, minimal centrilobular hypertrophy was noted in seven animals (statistically significant). Also in the liver, the incidence of focal cystic degeneration and bile duct hyperplasia was statistically significantly increased at 8000 ppm. Test article-related findings in the liver of females were limited to minimal to mild centrilobular hepatocellular hypertrophy at 2000 and 8000 ppm, and individual hepatocyte necrosis at 8000 ppm. In the testes of males there was a statistically significant increase in incidence of interstitial cell hyperplasia at 8000 ppm. In the lung, there was a test article-related increase in incidence (statistically significant) and severity of alveolar histiocytosis in females at 2000 and 8000 ppm and a test article-related increase in severity only in males at 8000 ppm. This finding, observed in both treated and control groups, represents an increase in the incidence and/or severity of a background lesion commonly observed in aging rats. Alveolar histiocytosis was not associated with any other test article-related finding in the lung. In the uterus of the 8000 ppm females, there was a statistically significant increase in changes typified by uterine dilatation, inflammation, and hyperplasia of endometrial epithelium and squamous cell hyperplasia in the cervix. In some instances the uterus was dilated with areas of inflammation and areas of hyperplasia of endometrial epithelium while in other instances there was cystic endometrial hyperplasia with a more generalized proliferation of endometrial epithelium.
Relevance of carcinogenic effects / potential:
At terminal necropsy, no statistically significant increase in any tumor type occurred in males.
In 8000 ppm females, a test article-related, statistically significant increase in squamous cell carcinoma of the uterus/cervix occurred which was statistically significant for both the Cochran-Armitage trend test and the Peto test and exceeded the historical control range. Squamous cell hyperplasia and endometrial proliferative changes were also present at 8000 ppm suggesting the increase in squamous cell carcinoma at 8000 ppm was test article related.
Key result
Dose descriptor:
NOAEL
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
food efficiency
haematology
clinical biochemistry
gross pathology
Remarks on result:
other: equivalent to 15.92 and 17.34 mg/kg body weight/day for male and female rats, respectively
Key result
Remarks on result:
other: The test substance is not oncogenic at dietary concentrations up to 8000 ppm for male rats and 2000 ppm for female rats.
Conclusions:
2 year NOAEL (chronic toxicity): 500 ppm (15.92 and 17.34 mg/kg body weight/day for male and female rats, respectively) (based on reductions in body weight and nutritional parameters, and clinical pathology and anatomic pathology findings at 2000 ppm and above)
The test substance is not oncogenic at dietary concentrations up to 8000 ppm for male rats and 2000 ppm for female rats.
Executive summary:

This study was conducted to evaluate the potential chronic toxicity and oncogenicity of the test article, when administered in the diet of rats for up to two years following the guidelines OECD 453 and U.S. EPA OPPTS 870.4300.

Five groups of 70 animals/sex/group received untreated diet or the test article orally via dietary admixture for up to 104 weeks at diet concentrations of 0, 100, 500, 2000, and 8000 ppm. Additional animals, designated for the 12-month interim necropsy (10 animals/sex/group), received the control or test diet for approximately 52 weeks.

Observations for morbidity, mortality, injury, and the availability of food and water were conducted for all animals twice daily through Week 52 and three times daily thereafter. Detailed clinical and mass observations were conducted on Day 1 prior to test article administration and weekly thereafter. Body weights were measured and recorded weekly during the study. Food consumption was measured and recorded weekly for the first 13 weeks of the study, then every other week thereafter. Body weight gain, food efficiency, and compound consumption were calculated. Ophthalmoscopic examinations were conducted pretest, at 12 months, and prior to the terminal necropsy. Blood and urine samples for clinical pathology evaluation were evaluated from designated animals at 3, 6, and 12 months. Blood smears for differential white blood cell (WBC) counts were collected from all animals at 12, 18, and 24 months and from all animals euthanized in extremis, and evaluated from all animals euthanized in extremis and from the control and high dose animals at the 24 month necropsy. At the end of the interim and terminal periods (including all animals found dead or euthanized in extremis), necropsy examinations were performed, organ weights were recorded, and tissues were collected. Tissues were microscopically examined for animals at 0 and 8000 ppm at the interim and terminal necropsies. The following tissues were considered to be possible target organs and were processed and examined from the 100, 500, and 2000 ppm groups at both the 12-month interim necropsy and the terminal necropsy: liver from all animals, testes from all males, lung (males at terminal necropsy only) and uterus (females at terminal necropsy only) from all females. An additional 25 animals/sex (sentinel animals) were co-housed with study animals, and serological health screens were conducted on these animals pretest and at 6, 12, 18, and 24 months.

The analytical results show that the test article was homogeneously mixed in the diet and present at the targeted concentrations. Test article was not detected in control samples.

The Week 1 to 51 mean daily intake values of the test substance for male rats in the 100, 500, 2000 and 8000 ppm dosing groups were 3.21, 16.35, 67.40, and 280.72 mg/kg body weight/day; the mean daily intake values for females in these dose groups were 3.96, 20.51, 89.14, and 413.76 mg/kg body weight/day. The overall (Week 1 to 104 for males and Week 1 to 103 for females) mean daily intake values of the test substance for male rats in the 100, 500, 2000 and 8000 ppm dosing groups for main study animals were 3.03, 15.92, 70.55, and 283.83 mg/kg body weight/day; the mean daily intake values for females in these dose groups were 3.23, 17.34, 73.80, and 395.88 mg/kg body weight/day.

No test article-related effects were noted on the following parameters: survival, cause of death, masses, ophthalmoscopic evaluations, differential WBC (white blood cell) counts, coagulation times, or urinalysis parameters. A greater number of females in the 8000 ppm group were noted to be thin in appearance relative to controls, which correlated with marked reductions in body weight that occurred in these animals.

Adverse, test article-related lower body weight and nutritional parameters were noted during the study in males and females at ≥2000 ppm. Effects observed in females were more severe than those observed in males exposed to the same dietary concentrations and the degree of change in absolute body weight (23.0% and 45.5% in 2000 and 8000 ppm females, respectively) were considered to have greatly exceeded a maximum tolerated dose for systemic toxicity. No adverse, test article-related effects on body weight or nutritional parameters were observed in males or females at 500 ppm and below.

Test article-related effects among hematology parameters for interim (12-month) animals included mild, non-adverse decreases in red cell mass and eosinophils in both sexes at 8000 ppm, with marginal effects on these parameters in animals receiving 2000 ppm. These findings lacked microscopic correlates and were not associated with increases in mean cell volume or reticulocyte count. Test article-related effects among clinical chemistry parameters for interim (12-month) animals included mild increases in urea nitrogen and phosphorus in both sexes at 8000 ppm, mild decreases in chloride in both sexes at 8000 ppm, mild increases in cholesterol in females at 2000 and 8000 ppm, and increases in bile acids in females at 8000 ppm. These findings were minor and non-adverse; they had no microscopic correlates.

No adverse test article-related macroscopic findings were noted following the one year interim necropsy. At this interim necropsy, test article-related increased mean liver weights occurred in both males and females at 2000 and 8000 ppm. At the terminal necropsy, liver weights were only statistically significantly increased in females at 2000 and 8000 ppm. The increased liver weights correlated to hepatocellular hypertrophy noted in both sexes at 8000 ppm at the interim and terminal necropsies and in females at 2000 ppm at the terminal necropsy. This change was likely the result of proliferation of microsomal enzymes within the hepatocytes as a result of test article administration and was not considered to be adverse. Interstitial cell hyperplasia of the testes was noted in males at 8000 ppm at the interim necropsy, but had no organ weight correlate.

Following the terminal necropsy, test article-related macroscopic observations were noted in the lung of male and female rats and the uterus of females. In the lung there was an increased incidence of focus/foci at 8000 ppm in both sexes and at 2000 ppm only in females, which correlated microscopically with alveolar histiocytosis. In the uterus of females there was an increased incidence of enlarged uterus at 2000 and 8000 ppm and mean uterus weights were increased at 8000 ppm. Enlarged uterus was correlative to the presence of tumors at 8000 ppm and dilatation of the uterus with or without additional findings of endometrial hyperplasia, inflammation, or hemorrhage.

At terminal necropsy, no statistically significant increase in any tumor type occurred in males.

In 8000 ppm females, a test article-related, statistically significant increase in squamous cell carcinoma of the uterus/cervix occurred which was statistically significant for both the Cochran-Armitage trend test and the Peto test and exceeded the historical control range. Squamous cell hyperplasia and endometrial proliferative changes were also present at 8000 ppm suggesting the increase in squamous cell carcinoma at 8000 ppm was test article related.

Test article-related non-neoplastic microscopic findings at terminal necropsy occurred in the liver, lung, and testes of males at 8000 ppm and in females in the liver at 2000 and 8000 ppm, and the lung and uterus at 8000 ppm. In the liver of males at 8000 ppm, minimal centrilobular hypertrophy was noted in seven animals (statistically significant). Also in the liver, the incidence of focal cystic degeneration and bile duct hyperplasia was statistically significantly increased at 8000 ppm. Test article-related findings in the liver of females were limited to minimal to mild centrilobular hepatocellular hypertrophy at 2000 and 8000 ppm, and individual hepatocyte necrosis at 8000 ppm. In the testes of males there was a statistically significant increase in incidence of interstitial cell hyperplasia at 8000 ppm. In the lung, there was a test article-related increase in incidence (statistically significant) and severity of alveolar histiocytosis in females at 2000 and 8000 ppm and a test article-related increase in severity only in males at 8000 ppm. This finding, observed in both treated and control groups, represents an increase in the incidence and/or severity of a background lesion commonly observed in aging rats. Alveolar histiocytosis was not associated with any other test article-related finding in the lung. In the uterus of the 8000 ppm females, there was a statistically significant increase in changes typified by uterine dilatation, inflammation, and hyperplasia of endometrial epithelium and squamous cell hyperplasia in the cervix. In some instances the uterus was dilated with areas of inflammation and areas of hyperplasia of endometrial epithelium while in other instances there was cystic endometrial hyperplasia with a more generalized proliferation of endometrial epithelium.

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for chronic toxicity of the test substance when administered in the diet for up to two years is 500 ppm, equivalent to 15.92 and 17.34 mg/kg body weight/day for male and female rats, respectively. The NOAEL is based on reductions in body weight and nutritional parameters, and clinical pathology and anatomic pathology findings at 2000 ppm and above. The test substance is not oncogenic at dietary concentrations up to 8000 ppm for male rats and 2000 ppm for female rats.

Test article-related neoplastic findings were limited to females at 8000 ppm and occurred at a dose level that produced marked reductions in absolute body weight that greatly exceeded the maximum tolerated dose for systemic toxicity.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

The chronic toxicity and carcinogenic potential of the test substance was assessed in a 2-year feeding study in rats and an 18-month feeding study in mice.

 

In rats, the NOAEL for chronic toxicity was 500 ppm in males and females (equivalent to 15.9 and 17.3 mg/kg bw/day, respectively). The primary findings in the study included marked reductions in body weight at dietary concentrations of 2000 and 8000 ppm; and based on the magnitude of the body weight effects at 8000 ppm (both sexes) and 2000 ppm (females), the changes were considered to have greatly exceeded the MTD. Increased liver weight and hepatocellular hypertrophy were observed in males and females and were consistent with non-adverse induction of liver metabolising enzymes that was noted in short-term feeding studies. In main study animals, increases in focal cystic degeneration and bile duct hyperplasia (males) and in individual cell necrosis (females) were observed in the liver at 8000 ppm. Interstitial cell hyperplasia was noted in high dose males at the interim and terminal examination time points. In the lung, there was an increase in incidence and/or severity of alveolar histiocytosis at 8000 ppm (both sexes) or 2000 ppm (females), a common background lesion in aging rats.

 

The test substance was not oncogenic in male rats. In the 8000 ppm females, an increase in squamous cell carcinoma of the uterus/cervix was observed. The incidences of this finding were 0/70, 0/70, 0/70, 1/70 (1.4%), and 5/70 (7.1%) at 0, 100, 500, 2000, and 8000 ppm. The increased incidence at 8000 ppm was statistically significant by the Cochran-Armitage trend test and exceeded the laboratory historical control range of 0-1.5%. In the uterus of 8000 ppm females, there were also statistically significant increases in changes typified by uterine dilatation, inflammation, and hyperplasia of the endometrial epithelium and an increase in squamous cell hyperplasia in the cervix. Therefore, the increase in uterine/cervix tumors in female rats at 8000 ppm was considered to be test substance related. A single occurrence of squamous cell carcinoma at 2000 ppm was not associated with an increase in squamous cell hyperplasia, was within the historical control range, and thus was not considered test-substance related.

 

The finding of uterine tumors in the 8000 ppm female rats is not considered relevant for humans based on the following lines of evidence:

 

            At this dietary concentration there were marked reductions in body weight that are considered to have greatly exceed the MTD. Absolute body weight and weight gain in the 8000 ppm females was 45.5 and 61.5% relative to controls, respectively. Typically, body weight changes greater than 10% are considered to exceed the MTD. Therefore, the biological relevance of toxicological findings is highly questionable at this dose level under conditions of severe body weight reductions indicative of marked systemic toxicity.

 

            Mechanistic studies have demonstrated that the test substance is not estrogenic which rules this out as a possible mechanism for the induction of uterine tumors in female rats. Negative results were obtained in an in vitro estrogen receptor binding assay and in an in vivo rat uterotrophic assay where the test substance did not induce increases in uterine weight that are characteristic of a direct estrogen agonist.

 

            Plasma prolactin levels were shown to decrease during the course of the rat uterotrophic assay at dose levels (300 and 500 mg/kg bw/day) similar to the corresponding diet concentration where the increase in uterine/cervix tumors was observed (8000 ppm, ~400 mg/kg bw/day). This finding suggests that the underlying mechanism in the induction of the tumors at this dose level is not relevant to humans. Prolactin is luteotrophic in rats in contrast to primates–it promotes progesterone production of the corpus luteum after ovulation and maintains gestation. Decreases in prolactin result in decreases in progesterone that in turn shift the estrogen:progesterone ratio towards estrogen dominance. Because prolactin is not luteotrophic in humans, this mechanism leading to uterine tumors in rats is not relevant to humans.

 

            A clear threshold was present as the increase in tumors of the uterus/cervix occurred only at the highest diet concentration of 8000 ppm (equivalent to ~400 mg/kg bw/day), which is 3333 times greater than the proposed acceptable daily intake (ADI) of 0.12 mg/kg bw/day, thereby providing a large margin of safety.

 

In mice, there were no treatment-related effects on body weight or any other in-life parameter in males or females administered the test substance up to and including a maximum dietary concentration of 7000 ppm for 18 months. In the liver there was an increase in the number of males with mass or nodule at 7000 ppm, an increase in liver weights in 7000 ppm males and in 2500 and 7000 ppm females, and an increase in hepatocellular hypertrophy in males at ≥ 200 ppm and in females at ≥ 2500 ppm. Enlarged spleen noted in 7000 ppm females correlated with increased spleen weights. An increase in severity/incidence of extramedullary hematopoiesis was observed in 7000 ppm males and ≥ 800 ppm females, but in the absence of correlative changes in bone marrow or other changes indicative of adverse effects on red blood cells, was considered to be non-adverse.

 

Hepatocellular adenomas were increased in the 7000 ppm male mice. The incidences of this finding were 9/60, 6/60, 4/60, 12/60, and 17/60 at 0, 200, 800, 2500, and 7000 ppm and was statistically significant at 7000 ppm by Cochran-Armitage trend test. These tumors are considered secondary to enzyme induction followed by a subsequent increase in cell proliferation in the liver, and not relevant to humans for the following reasons:

 

            The primary findings in short-term (28- and 90-day) feeding studies in mice were increases in liver weight accompanied by hepatocellular hypertrophy. These changes were more prominent in male mice compared to female mice.

 

            The changes in liver weight and hepatocellular hypertrophy were consistent with the observed induction of hepatic metabolizing enzymes in the 28-day mouse study where increases in total hepatic microsomal cytochrome P450 enzyme content and cytochrome isozymes Cyp4A1/2/3 and Cyp2E1 were noted in both males and females. In males, Cyp2B1/2 was also selectively increased whereas Cyp1A2 was increased in females.

 

            In a 28-day liver mechanistic study conducted in male mice at 0, 200, 800, 2500 and 7000 ppm, exposure to 7000 ppm resulted in pronounced increases in Cyp2B10 gene expression consistent with activation of the constitutive androstane receptor (CAR), a temporary increase in cell proliferation, hepatocellular hypertrophy, and increased liver weight. These changes were consistent with those of the prototypical CAR inducer phenobarbital (PB) included in the study as a positive control. Thus, the findings support a phenobarbital-like mechanism for liver tumor induction in the 7000 ppm male mice which is considered not plausible for humans (Elcombe et al., (2014) Critical Reviews in Toxicology, 44(1):64-82).

 

            While many studies have shown that PB (and/or its sodium salt) can act as a non-genotoxic carcinogen and tumor promoter in rats and mice, it does not appear to produce liver tumors in humans. PB has been used as a sedative, hypnotic and antiepileptic drug in humans for many years, and a number of epidemiological studies have concluded that there is no evidence for a specific role of PB in human liver cancer risk. Therefore, compounds that cause rat or mouse liver tumors through a CAR-mediated mechanism are not expected to increase the risk of liver tumor development in humans (Elcombe et al., 2014).

 

            In neither the short-term mouse studies or in the 18-month mouse study is there evidence of cytotoxicity to the liver which rules this out as a possible mechanism for the induction of liver adenomas in male mice.

 

            Liver adenomas in high dose male mice occurred at a dose level (727 mg/kg bw/day) at least

6000 times greater than the proposed ADI of 0.12 mg/kg bw/day thereby providing a very large margin of safety.

 

The overall weight of evidence indicates that the test substance does not induce gene mutations or chromosome aberrations and therefore can be considered as nongenotoxic. Although tumors of the uterus/cervix occurred in high dose female rats and benign liver adenomas occurred in high dose male mice in the long-term chronic and carcinogenicity studies conducted with the test substance, these findings are of either no biological relevance as they occurred at a dose level that greatly exceeded the MTD (rats) and/or are due to mechanisms specific to rodents that are not relevant to humans. Therefore, the test substance does not pose a carcinogenic concern.

Additional information

Justification for classification or non-classification

Based on the results of chronic feeding studies in rats and mice, the findings are of either no biological relevance as they occurred at a dose level that greatly exceeded the MTD and/or are due to mechanisms specific to rodents that are not relevant to humans and thus it can be concluded that the test substance does not pose a carcinogenic concern for humans. Therefore, the test substance is not classified for carcinogenicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.