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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

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Endpoint:
carcinogenicity: 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 451 (Carcinogenicity Studies)
Deviations:
yes
Remarks:
Detailed clinical observations were not performed on Weeks 78 and 79 in error prior to necropsy for all animals. Since animals were observed daily at least twice, not performing detailed clinical observation does not impact the study.
GLP compliance:
yes
Specific details on test material used for the study:
99.3% purity
Species:
mouse
Strain:
other: Crlj:CD1(ICR)
Details on species / strain selection:
The Crlj:CD1(ICR) mouse was selected based on consistently acceptable health status and on experience with this strain at the test facility.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: 28.6-38.6 g (males); 22.2-30.0 g (females)
- Housing: One male/cage and five females/cage were housed in solid bottom cages during acclimatization. Two females/cage were housed in solid bottom cage during the pre-treatment and treatment periods. Males were housed individually in solid bottom cages during the pre-treatment and treatment periods. The cages of animals fed different dietary concentration were located in separate racks and the cage racks were relocated within the animal room every four weeks. Cages were also rotated among different shelves on each rack on the same schedule.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±3°C
- Humidity (%): 50±10%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hour light and 12 hour dark
Route of administration:
oral: feed
Vehicle:
other: Lab Diet® #5002
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of the test substance were performed by high-performance liquid chromatography with UV detection. The linearity of the standard curve was examined by injection of a series of 5 standard solutions that encompass the working range (2.5-25 µg/mL) around the concentration of the single point calibration standard at 10 µg/mL. The response factor (Concentration peak area) calculated for each standard must be within 5% of the mean response factor for all standards. The reproducibility of the injection was evaluated on six replicate injections of the lowest (2.5 µg/mL) and the highest (25 µg/mL) standard solutions. The Coefficient variation (%CV) obtained for the six peak responses from the six replicate injections should be less than 2%. The accuracy and precision of the method was determined by preparing six procedural recoveries containing test substances at the intended lowest (100 ppm) and highest (4800 ppm) concentrations and analyzing these in accordance with the following analytical procedure: the accuracy of the method was calculated by measuring the analyzed concentration relative to the spiked concentration. The minimum criterion for acceptable mean accuracy is 100 ± 10%. Precision was expressed as the value for the percent coefficient of variation (%CV) of the analytical recovery values, which is expected to be less than 10.0%.

Duplicate samples of all dietary concentrations were collected at the initial diet preparation and analyzed to verify the concentration (average of homogeneity samples) and homogeneity of the test substance in the diets. Duplicate samples were taken from the 100 and 4800 ppm concentration of the initial diet preparation and analyzed to verify the stability of the test substance in the diet. Among duplicate samples, one sample was analyzed and the other was stored frozen for backup. Diet samples were collected from all concentrations every 3 months to verify concentration. All remaining samples were discarded after satisfactory analytical results.

For the homogeneity at the 100 ppm concentration of the second diet preparation, the analysis of back up samples was performed with single sample instead of triplicate samples. This deviation was considered unlikely to affect the integrity of the study since reanalysis of back up samples was done with triplicate samples.

The 100 ppm diet with below acceptable analysis results was given to the 100 ppm animals for 1 day (test day 175). This deviation occurred temporarily and therefore was considered unlikely to affect the integrity of the study.

The control diet was analyzed each time.
Duration of treatment / exposure:
18 months
Frequency of treatment:
Daily
Post exposure period:
No
Dose / conc.:
100 ppm
Dose / conc.:
600 ppm
Dose / conc.:
2 400 ppm
Dose / conc.:
4 800 ppm
No. of animals per sex per dose:
60
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Doses were selected by the Sponsor based on the results of previous toxicity studies with the test substance.
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cage-site examinations to detect moribund or dead mice (including spares during the first two weeks) were conducted once daily in the morning throughout the study except during the acclimation period. The animals were observed more than once when they were in poor condition. Additional cage-site evaluations to detect acute clinical signs of systemic toxicity, abnormal behavior and/or appearance among mice were conducted once a day, except on the days when detailed clinical observations were conducted.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each mouse was individually handled and examined for abnormal behavior and appearance three times in the pre-treatment period (including spare animal), at the first day of dosing prior to test article exposure and once a week thereafter. Detailed clinical observations in a standardized arena were also evaluated on all mice. The detailed clinical observations included (but were not limited to) evaluation of fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, and unusual respiratory pattern), changes in gait, posture, response to handling, presence of clonic, tonic, stereotypical, or bizarre behavior and presence of masses etc. Any abnormal clinical sign noted was recorded weekly up to Week 77 for all animals.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was measured twice during acclimation period (Days -17 and -12) and once during pre-treatment period (day -7). During the treatment period, all animals were weighed on Day 0 prior to test article exposure, at the first day of every study week during the first 13 weeks, every other week thereafter and stopped at the last biweekly weight date prior to necropsy. On the necropsy day, animals were weighed prior to necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; Individual food consumption of all animals was calculated during pre-treatment (Week -1) and weekly during the first 13 weeks and every other week thereafter. Recording of food consumption was stopped at the last biweekly measurement prior to the necropsy for all animals. The test article containing diets was given to the animals until the necropsy.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY: Yes; calculated from food consumption and body weight data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes; Ophthalmological examination on all animals was conducted once during the pre-treatment period (including spare animals) and at the end of study. All surviving mice were examined prior to the final sacrifice. Both eyes of each mouse were examined by focal illumination and indirect ophthalmoscope after mydriasis had been produced with Mydrin-P.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 12 and 18 months
- Anaesthetic used for blood collection: Yes; isoflurane
- Animals fasted: Yes
- How many animals: All
- Parameters examined: Blood smears were prepared for evaluation of differential WBC counts from all mice sacrificed in extremis and from all mice at 12 and 18 months. Differential WBC counts were evaluated in all mice sacrificed in extremis and in control and high-concentration mice from the necropsy. Blood smears were prepared at the time of blood collection and were stained with Wright-Giemsa stain for microscopic evaluation. The analysis was performed only at control and high concentration animals at 18 months and the
results did not warrant analysis in other groups.

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; see Table 1
HISTOPATHOLOGY: Yes; see Table 1


Complete necropsy examinations were performed under the direct supervision of veterinary
pathologists on all animals found dead (tissue integrity permitting), accidentally killed,
euthanized in extremis, and euthanized at scheduled necropsy (test days 546 to 550 for males and 548 to557 for females, respectively). No necropsy was performed on spare animals. The animals were examined carefully for external abnormalities. The abdominal, thoracic and cranial cavities were examined for abnormalities and the organs were removed and examined. All tissues from each animal were preserved in 10% neutral buffered formalin with the following exceptions: the eyes (including retina and optic nerve) were fixed in Davidson’s fixative; and the testes and epididymides were fixed in Bouin’s fixative for approximately 24 hours, then transferred to 70% ethanol. Formalin was infused into the lung via the trachea and into the urinary bladder. Relative organ weights (relative to terminal body weight; relative to brain weight) were calculated. Terminal body weights determined just prior to necropsy were used in the assessment of organ weight changes. Organs from mice found dead, sacrificed in extremis, or accidentally killed were not weighed.

All tissues collected from mice at the 18-month time point, in the high-concentration and control groups, and from mice that were found dead or accidentally killed (tissue integrity permitting), or were sacrificed in extremis, were further processed to slides, stained with hematoxylin and eosin, and examined microscopically. In the 100, 600 and 2400 ppm groups, gross lesions for both sexes, liver, duodenum and stomach for males, and liver for females were processed to slides and stained for microscopic examination.
Statistics:
Body Weight, Body Weight Gain, Food Consumption, Food Efficiency, Clinical Pathology, Incidence of Ophthalmology, Observations and Organ Weight: Preliminary Test: Bartlett’s test for homogeneity; If preliminary test is not significant: One-way analysis of variance followed by
Dunnett’s test and If preliminary test is significant: Kruskal-Wallis test followed by Dunn’s Rank Sum test

Survival: Cochran-Armitage test for trend and Kaplan-Meier analysis

Incidence of Microscopic Lesions: Cochran-Armitage test for trend and Fisher’s exact test

Statistical analyses were performed by comparing the different dose groups with the vehicle control group using Path/Tox System (ver. 4.2.2, Xybion Medical Systems Corporation, USA) and/or Statistical Analysis Systems (SAS/STAT Version 9.1.3. or 9.2, Cary, NC, USA). Statistical analyses for survival and microscopic findings were performed by the Sponsor. Because of significant difference in survival in high-dose male mice as compared to controls at the end of the study, survival adjusted analysis (Peto analysis and Poly-3 test) were used to compare microscopic findings in the liver and duodenum in males. The Poly-3 test, a modified Cochran-Armitage linear trend test that adjusts for survival, is commonly used by United States National Toxicology Program (NTP) to analyze tumor incidences in rodent carcinogenicity studies when there is a survival difference.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical signs such as thin appearance, subdued behaviour, clonic convulsion, irregular respiration, loss of fur, paleness, scratch wound, hard skin, lacrimation, cloud eyes, and abdominal distention were observed at all concentrations (including controls) sporadically during the treatment period.
Since there were no dose response, none of these are considered test article-related.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There was a statistically significant increase in survival at terminal sacrifice in the 2400 and
4800 ppm male groups compared to control. This statistically significant increase in survival was
considered spurious.

2400 ppm: 83% survival in males and 72% in females versus 72% in male controls and 78% in female controls
4800 ppm: 93% survival in males and 75% in females versus 72% in male controls and 78% in female controls
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant changes were observed in body weights and body weight gains sporadically during treatment period. Mean final (test day 539) body weight and overall (test day 0-539) body weight gain in 4800 ppm males were 97% and 92%, respectively, of controls. Mean final (test day 539) body weight and overall (test day 0-539) body weight gain in 4800 ppm females were 94% and 86%, respectively, of controls. None of these changes were statistically significant compared to controls.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant changes were observed in food consumption and food efficiency sporadically during treatment period. Mean overall (test day 0-539) food consumption and food efficiency in 4800 ppm males were 100% and 100%, respectively, of controls. Mean overall (test day 0-539) food consumption and food efficiency in 4800 ppm females were 101.9% and 85.7%, respectively, of controls. None of these changes
were statistically significant compared to controls.

In males, overall (days 0-539) mean daily intake of the test article in the 0, 100, 600, 2400, and 4800 ppm groups was 0, 12.1, 70.8, 293.3, and 583.2 mg/kg body weight/day, respectively. In females, overall (days 0-539) mean daily intake of the test article in the 0, 100, 600, 2400, and 4800 ppm groups was 0, 16.4, 98.6, 411.5, and 799.3 mg/kg body weight/day, respectively.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant changes were observed in food consumption and food efficiency sporadically during treatment period. Mean overall (test day 0-539) food consumption and food efficiency in 4800 ppm males were 100% and 100%, respectively, of controls. Mean overall (test day 0-539) food consumption and food efficiency in 4800 ppm females were 101.9% and 85.7%, respectively, of controls. None of these changes were statistically significant compared to controls.
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related changes in white blood cell relative differential counts. The following statistically significant changes at study termination in mean white blood cell differential counts were considered to be unrelated to treatment and thus non-adverse. Neutrophils were decreased and lymphocytes were increased at study termination in females fed 4800 ppm, compared to controls. Because these 2 cell types comprise the majority of circulating leukocytes, changes in relative counts in one of these cell types is reflected by changes in the other. Neutrophil counts were 44% and 37%, and lymphocyte counts were 53% and 61% for females fed 0 and 4800 ppm, respectively. Because differential counts for older mice are quite variable, the standard deviations for these means were large (all standard deviations were about 15). Although the mean neutrophil count was lower in 4800 ppm females than in controls, the upper limit of the range of individual values was higher in 4800 ppm mice compared to controls. Likewise, although the mean lymphocyte count was higher in 4800 ppm females compared to controls, the lower limit of the range of individual values was lower in 4800 ppm females. These changes in mean neutrophils and lymphocytes counts were considered to be normal aging variation and unrelated to treatment and non-adverse.
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Clinical signs such as subdued behavior were observed at all concentrations (including controls) sporadically during the treatment period.
Since there were no dose response, none of these are considered test article-related.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Following 18 months of dietary exposure, a test article-related but non-adverse increase in liver weights was observed in males and females fed 2400 or 4800 ppm of the test substance. In males exposed to 4800 ppm of the test article, mean absolute and relative (% body weight and % brain weight) liver weights were increased approximately 20%, 22% and 20%, respectively as compared to control. The differences in mean absolute and mean relative (% body weight) liver weights in 4800 ppm were statistically significant (p <0.05). In 2400 ppm males, mean absolute and relative (% body weight and % brain weight) liver weights were increased approximately 13%, 16% and 12%, respectively. Although none of the increases in these liver weight parameters in 2400 ppm males were statistically significant, these weight increases were considered test article related.

In females fed the 4800 ppm diet, mean absolute and relative (% body and % brain weight) liver weights were increased approximately 25%, 31% and 26%, respectively compared to control (variable statistical significance). In females fed 2400 ppm, mean absolute and relative (% body
and brain weight) liver weights were increased 23%, 26% and 21%, respectively compared to control. Only the difference in mean relative (% body weight) liver weight was statistically significant (p <0.01) in 2400 ppm females. The liver weight changes at both 2400 and 4800 ppm
were considered test article related.

The liver weight increases noted above in males were not associated with detectable microscopic hepatocellular hypertrophy. Liver weight effects in females correlated with the microscopic finding of hepatocellular hypertrophy at the same exposure levels (see microscopic findings).
These changes in liver weight parameters were not associated with other microscopic findings indicative of liver toxicity. Therefore, these liver weight changes considered to represent an adaptive response associated with metabolism of the test-substance and thus were not considered
adverse.

In females, mean relative (% body weight) adrenal weight was increased (variable statistical significant) approximately 19%, 13%, 16% and 17% compared to controls in the 100, 600, 2400 and 4800 ppm dietary exposure groups respectively. These adrenal weight changes were not dose related, were not associated with changes in mean absolute adrenal weight or adrenal weight relative to brain weight, and there were no correlative microscopic findings in the adrenal gland. In addition, no adrenal weight changes occurred in male mice at any concentration. Therefore, this change in mean relative (% body weight) adrenal weights was considered spurious and unrelated to dietary exposure to the test article.

In females, mean relative (% body weight) kidney weight was increased approximately 10% and 9% compared to control in the 2400 and 4800 ppm groups, respectively (statistically significant). However, this was not considered test article related for the following reasons: higher relative kidney weight did not occur in a dose related manner, the mean absolute and relative kidney weights (% brain weight) were not statistically significantly increased, male kidney weight values were not affected, and there was no associated gross or microscopic kidney pathology.

In females, mean relative (% body weight) heart weight was increased (statistically significant) approximately 9%, 10%, 14% and 7% compared to controls in the 100, 600, 2400 and 4800 ppm dietary exposure groups respectively. These heart weight changes were not dose related, were not associated with changes in mean absolute heart weight or heart weight relative to brain weight, and there were no correlative microscopic findings in the heart. In addition, no treatment-related heart weight changes occurred in male mice at any concentration. Therefore, this change in mean relative (% body weight) heart weights was considered spurious and unrelated to dietary exposure to the test article.

All other individual and mean organ weight differences were interpreted as spurious and unrelated to dietary exposure of test article.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In male mice, the incidence of liver masses/nodules noted at necropsy was increased at 2400 ppm (27 of 60 affected) and 4800 ppm (29 of 60 affected) relative to controls (20 of 60 affected). In both control and treated male mice, gross liver masses/nodules primarily correlated microscopically to hepatocellular adenomas/carcinomas or hemangiosarcomas, neither of which was considered to be test article related findings (see microscopic findings).

Grossly, a decrease in the incidence of ‘enlarged’ mandibular lymph node and thymus was observed in 4800 ppm males (2 of 60 affected) as compared to controls (7 of 60 affected). A similar decrease in gross incidence of ‘enlarged’ thymus was observed in 4800 ppm males (1 of 60 affected) as compared to controls (5 of 60 affected). Microscopically, ‘enlarged’ mandibular lymph node and thymus mostly correlated with malignant lymphoma, a systemic neoplasm. Although there was a decrease in number of 4800 male mice having malignant lymphoma, this was not interpreted as a test-substance related effect. Thus, decrease in the incidence of ‘enlarged’ mandibular lymph node and thymus was considered unrelated to treatment.

An increase in the incidence of ‘enlarged’ seminal vesicle was observed in the treated males as compared to the controls (14/60, 25/60, 27/60, 24/60, 22/60 in 0, 100, 600, 2400, and 4800 ppm, respectively). Microscopically, this gross finding was only confirmed in few animals across all the groups as ‘dilatation of seminal vesicle’ (often grossly enlarged seminal vesicle was unremarkable on microscopic examination). Since microscopic data did not demonstrate test article-related effect in seminal vesicle dilatation, the increased incidence of this gross finding in seminal vesicles was not considered test-substance related.

A marginal increase in the incidence of kidney cysts in the 4800 ppm males (13 of 60 affected) versus the controls (6 of 60 affected) was also not considered test article related since this apparent gross trend was not confirmed by either organ weight data or microscopic findings. An increase in the incidence of gross uterus/cervix dilatation was observed in the treated females compared to the controls (19/60, 25/60, 20/60, 27/60, 27/60 in 0, 100, 600, 2400, and 4800 ppm, respectively). The gross term “uterus/cervix dilatation” is non-specific. This gross observation most often microscopically correlated with cystic endometrial hyperplasia, endometrial polyp smooth muscle hyperplasia, angiectasis, leiomyosarcoma, histiocytic sarcoma and stromal sarcoma. None of these microscopic findings in uterus or cervix was interpreted as test-substance related. Therefore, increased incidence of uterus/cervix dilatation in 2400 ppm and 4800 ppm females were considered spurious and not test article-related.

All other gross observations noted in this study were considered to be common age-related changes in the mice of this age and strain and the incidence of such gross observations were considered to be with in range of normal biologic variation of this species.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test article-related microscopic findings (non-neoplastic)were present in the duodenum and liver.

In males, an increased incidence and severity of duodenal mucosal hyperplasia was present in the 2400 and 4800 ppm groups compared to control. This increase was statistically significant by the Cochran-Armitage trend test; p<0.05 in the 4800 ppm male group only. A pairwise comparison by Fischer’s exact test, as well as survival adjusted statistics (Poly-3 test and Peto analysis), showed no significant difference in the incidence of duodenal hyperplasia in 2400 ppm and 4800 ppm males as compared to controls. Twenty-one of the 23 males with duodenal mucosal hyperplasia were among the terminal sacrifice mice. Nevertheless, based on the dose response for severity grade of this lesion, duodenal hyperplasia was considered a test article-related and adverse finding in the 2400 and 4800 ppm males. A similar test article-related finding was not observed in females at any of the dietary concentrations evaluated.

Microscopically, duodenal hyperplasia was characterized by thickened areas of duodenal mucosa with irregular luminal borders and increased basophilic staining compared to normal mucosa. These thickened mucosal areas consisted of densely arranged crypts and tubular glands lined by proliferating epithelium. In more severely affected animals, duodenal hyperplasia was often associated with dilation of mucosal glands. There was no evidence of invasion of proliferating epithelium into underlying submucosa.

In the liver, compound-related microscopic findings consisted of increased incidences of hepatocellular hypertrophy in females fed ≥ 2400 ppm and mixed foci of cellular alterations in males at 4800 ppm.

In female mice, centrilobular hepatocellular hypertrophy was present in 2/58, 0/59, 3/60, 5/60, and 28/60 mice given 0, 100, 600, 2400 or 4800 ppm, respectively. All were graded as minimal (grade 1 of 4) except for two 4800 ppm female mice, which were graded as mild (grade 2 of 4). Thirty-four of the 38 females with hepatocellular hypertrophy were among the terminal sacrifice mice. Hepatocellular hypertrophy can be observed in aging mice as spontaneous background finding. In this study, a low incidence was observed in both male and female control mice. An incidence of 3/60 minimal hepatocellular hypertrophy in 600 ppm females was not considered biologically different from control females and therefore was considered unrelated to treatment. However, the dose related increased incidences and degree of hepatocellular hypertrophy in the 2400 and 4800 ppm female mice (incidences were only statistically significant in the 4800 ppm group), were interpreted to be test article related.

The hepatocellular hypertrophy diagnosed in this study was characterized by enlarged centrilobular hepatocytes. The enlargement was due to an increase in cytoplasmic volume rather than nuclear size. The increase in the incidence and degree of centrilobular hepatocellular hypertrophy, as observed in this study, was consistent with the induction of hepatocellular enzyme systems secondary to xenobiotic exposure and was not associated with microscopic evidence of hepatotoxicity. Therefore, hepatocellular hypertrophy was considered adaptive and non-adverse.

Treatment related hepatocellular hypertrophy was not observed in male mice at any concentration.

The incidence of hepatic adenoma and combined hepatic adenoma/carcinoma (‘Animals with at least one hepatocelluar neoplasm’) was higher in the 4800 ppm male group compared to control but was considered to be the result of increased survival in this group based on the following:
• Although statistically significant by the Cochran-Armitage trend test, these differences were not statistically significant by the Fisher’s exact test or when adjusted for survival by either the Poly-3 test or Peto analysis.
• There was no apparent increase in tumor multiplicity in this group.
• There were no statistically significant differences in the incidence of hepatic carcinoma in the 4800 ppm male group compared to control and thus, there was no apparent test article related progression of adenomas to carcinomas.

Therefore, the higher incidences of hepatocellular neoplasms in 4800 ppm males was not considered to be test article related, but rather was considered secondary to the increased survival in the 4800 ppm male group (93% compared to 72% in control). The incidences of spontaneous
liver neoplasms in most strains of mice are expected to increase with increased survival.

As with liver adenomas, the incidences of mixed foci of cellular alteration were also higher in the 4800 ppm male and were significantly increased by the Cochran Armitage trend test but not by Fisher’s exact test or when adjusted for survival by Poly-3 test or Peto analysis. Therefore, as
with adenomas, this difference was considered to be secondary to increased survival in this group and not test article related.

In addition to the liver and duodenum (male only), stomach was processed and examined in all of the intermediate-concentration male mice (Groups 2, 3, and 4) in order to clarify difference in the incidence of glandular hyperplasia between the control and high-concentration groups. In
males, stomach glandular hyperplasia was present in 10/53, 22/58, 22/58, 26/59 and 18/58 mice given 0, 100, 600, 2400, or 4800 ppm, respectively. The incidence of stomach glandular hyperplasia was statistically increased (Fischer’s exact test; p<0.05) in the 2400 ppm males.
Glandular hyperplasia is a common background lesion in aging mice and incidences of this finding in the current study were comparable to other studies performed in similar lab and to published studies. In the present study, stomach glandular hyperplasia was present in males in all groups (including controls) and the incidence and severity of this lesion was not increased in a clear dose-related manner at the higher dietary concentrations (26/59 at 2400 ppm vs. 18/59 at 4800 ppm). In addition, there was no statistically significant difference in the incidence of stomach glandular hyperplasia in female mice (actually there incidence of this lesion was lower in high-dose females as compared to control females - 10/57 and 4/60 in controls and 4800 ppm females respectively). Therefore, increased incidence of stomach glandular hyperplasia in 2400 ppm males was interpreted as spurious and unrelated to treatment.

All other statistically significant differences between treated groups and the respective controls were not considered to be test article-related. These did not occur in a dose-related manner or were decreased relative to controls and represent findings commonly seen in the aging mice of this strain.
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
600 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
4 800 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effects observed at highest dose tested
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
2 400 ppm
System:
gastrointestinal tract
Organ:
duodenum
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Conclusions:
Under the conditions of this study, the test substance is not oncogenic in male or female mice following 18 months of dietry adminstration.

Test article-related and biologically adverse microscopic findings were limited to non-neoplastic effect in the duodenum and consisted of increased incidences and degree of mucosal hyperplasia in males at 2400 ppm and above.
Executive summary:

Five groups of young adult male and female Crlj:CD1(ICR) mice (60/sex/group) were administered diets that contained 0, 100, 600, 2400, or 4800 ppm of the test substance for approximately 18 months. Samples of the diets were analyzed and demonstrated homogeneity, stability, and targeted concentrations of the test article in the diet. Body weights and food consumption were evaluated weekly for the first 13 weeks, then every other week thereafter. Detailed clinical observations were evaluated weekly. Ophthalmological assessments were performed prior to the start of dietary exposure and near the end of the exposure period. White blood cell differential counts were evaluated in surviving mice at the end of the exposure period and in mice that were sacrificed in extremis. After approximately 18 months of dietary exposure, mice were sacrificed and given a gross and microscopic pathological examination.

The overall mean daily intakes of the test substance in the 0, 100, 600, 2400, and 4800 ppm male groups were approximately 0, 12, 71, 293, and 583 mg/kg/day, respectively. In females, mean daily intakes were approximately 0, 16, 99, 412, and 799 mg/kg/day, respectively.

No test article-related changes were observed in the following observations in male and female mice fed up to 4800 ppm of the test substance: clinical observation, body weight parameters, food intake parameters, ophthalmology, white blood cell differential counts, cause of death, and gross pathological parameters, and neoplastic changes.

Test article-related and biologically adverse microscopic findings were limited to non-neoplastic effects in the duodenum and consisted of increased incidences and severity of mucosal hyperplasia in males fed dietary concentrations of 2400 or 4800 ppm. Female mice did not exhibit treatment-related duodenal mucosal hyperplasia at any concentration.

Test article-related (non-adverse) increases in liver weights were observed in males and females fed dietary concentrations of ≥2400 ppm. In female mice, the increased liver weights correlated with the test article-related microscopic finding of hepatocellular hypertrophy. Both the liver weight increases and hepatocellular hypertrophy were consistent with hepatic enzyme induction and were interpreted to be not adverse.

The test substance is not an oncogen in mice. Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) was 600 ppm for male mice and 4800 ppm for female mice, equivalent to 71 and 799 mg/kg/day, respectively.

Endpoint:
carcinogenicity: 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 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
yes
Remarks:
See the "Any other information on materials and methods" section for the deviations.
GLP compliance:
yes
Specific details on test material used for the study:
99.3% purity
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 administered via the oral route and for which there is a large historical database.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: 244-291 g (males); 178-219 g (females)
- Housing: The animals were individually housed in suspended, stainless steel, wire-mesh type cages in an environmentally controlled room. Beginning in Week 88, all animals were housed in solid bottom caging with non-aromatic bedding to reduce development of foot and joint trauma effects. Nylabones were provided to all animals beginning on Day -7.
- Diet (e.g. ad libitum): Approximately 200 to 300 g of diet was offered to each animal on a weekly basis, except during designated periods
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26°C (64-79°F)
- Humidity (%): 30-70%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hour light and 12 hour dark
Route of administration:
oral: feed
Vehicle:
other: Meal Lab Diet® Certified Rodent Diet #5002
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were shipped and stored at room temperature to the MPI Research, Inc., site at State College, Pennsylvania, for analysis. Backup samples were shipped on dry ice as needed. All analytical work was conducted by MPI Research, Inc., using a high-performance liquid chromatographic (HPLC) analytical method validated under MPI Research Study Number 125-094. Deviations from the Analytical Method are as follows:

In data set P4129_070308, samples were placed in ice water after sonication.

In data set P4129_070909, to ensure proper mixing, homogeneity samples were ground in a grinder before aliquots were collected for analysis.
Duration of treatment / exposure:
2 years
Frequency of treatment:
Daily
Post exposure period:
No
Dose / conc.:
50 ppm
Dose / conc.:
200 ppm
Dose / conc.:
1 000 ppm
Dose / conc.:
3 500 ppm
No. of animals per sex per dose:
80
Control animals:
yes, plain diet
Details on study design:
Five groups of 80 animals/sex/group received untreated diet or the test article orally via dietary admixture approximately 105 weeks (males) or 103 weeks (females) at diet concentrations of 0, 50, 200, 1000, and 3500 PPM. Following 12 months of treatment, ten animals/sex/group were submitted to an interim necropsy.
Positive control:
No
Observations and examinations performed and frequency:
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 weekly. Body weights were measured and recorded weekly from Weeks -1 to 13 and every fourth week thereafter. Food consumption was measured and recorded weekly from Weeks -1 to 13, every fourth week from Weeks 17 to 85, and weekly beginning on Week 88. Body weight gain, food efficiency, and compound consumption were calculated.
Sacrifice and pathology:
Necropsy examinations were performed, organ weights were recorded, and selected tissues (see below) were collected for microscopic examination from 10 animals/sex/group at the interim necropsy on Day 365 and from all remaining animals at the terminal necropsy. Serological health screens were conducted on five unassigned animals/sex pretest and on an additional 15 animals/sex co-housed with study animals at 6, 12, and 18 months.

The following tissues were examined: Adrenal gland, Aorta, Bone marrow smear, Bone with bone marrow, femur, Bone with bone marrow, sternum, Brain (cerebrum, midbrain, cerebellum, medulla/pons), Coagulating gland, Epididymis, Esophagus, Eye (with optic nerve and retina), Heart, Joint, tibiofemoral, Kidney, Large intestine, cecum, Large intestine, colon, Large intestine, rectum, Larynx, Liver, Lung, Lymph node, mandibular, Lymph node, mesenteric, Mammary gland (process females only), Nose (four sections), Nerve, sciatic, Ovary, Pancreas, Peyers Patch, Pharynx, Pituitary, Prostate, Salivary gland, mandibular, Salivary gland, parotid, Salivary gland, sublingual, Seminal vesicle, Skeletal muscle, biceps femoris, Skin, Small intestine, duodenum, Small intestine, ileum, Small intestine, jejunum, Spinal cord, cervical, Spinal cord, lumbar, Spinal cord, thoracic, Spleen, Stomach, glandular, Stomach, nonglandular, Testis, Thymus, Thyroid (with parathyroid), Trachea, Urinary bladder, Uterus with cervix, Vagina, Gross lesions, and Tissue masses with regional lymph node.
Other examinations:
Ophthalmoscopic examinations were conducted pretest and prior to the interim and terminal necropsy. Blood and urine samples for clinical pathology evaluations were collected from designated animals at 3, 6, and 12 months and from all surviving animals at the terminal necropsy (hematology only).
Statistics:
See the "Any other information on materials and methods" section for details on the Statistics used in this study.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There was an increase in the incidence of soft faeces at 1000 and 3500 ppm primarily in males that was considered possibly test article-related but not adverse.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Survival in the 3500 ppm male and female groups and in the 1000 ppm female group was significantly greater than in controls. This increase is likely due to lower body weight in these groups (not statistically significant in females at 1000 ppm).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight and body weight gain were reduced during the study in both sexes at 3500 ppm. In males and females, mean body weight was 9% and 17% below control, respectively, at Week 49, and 11% and 17% below control, respectively, on the last weigh day before final sacrifice. Mean body weight gain in this group was 15% and 33% below control for males and females, respectively, over weeks 1 to 49, and 18% and 28% below control, respectively, over the two year exposure period. All of these differences were statistically significant except the male final body weight and overall body weight gain. However, these values were significantly different from control for most of the study. These body weight findings were associated with significantly lower mean food consumption and food efficiency over the first year at this exposure level which continued for the duration of the study (variable statistical significance). Body weight and nutritional parameters in lower
concentration groups were generally comparable to control over the study.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Body weight findings were associated with significantly lower mean food consumption and food efficiency over the first year in the 3500 ppm group which continued for the duration of the study (variable statistical significance). Body weight and nutritional parameters in lower concentration groups were generally comparable to control over the study.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Body weight findings were associated with significantly lower mean food consumption and food efficiency over the first year in the 3500 ppm group which continued for the duration of the study (variable statistical significance). Body weight and nutritional parameters in lower concentration groups were generally comparable to control over the study.
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
1-year Interim

There were no test article-related effects on haematology parameters through 12 months of treatment. Sporadic statistical differences were observed that were considered not meaningful due to the magnitude or direction of change, lack of dose response, or lack of consistency over time. Unscheduled samples (i.e., special collections) were collected from several animals across all groups, including controls prior to euthanasia in extremis. There was no dose-related pattern of changes to suggest test article-related effects in these animals.

2-year Terminal

No test article-related effects on haematology parameters were identified in either sex at any concentration at any study interval. There were infrequent statistically significant alterations in mean values among various haematology parameters that were not considered relevant to the test article due to their small magnitude, direction of change, sporadic nature, and/or lack of a dose response. Occasional individual animals from all dose groups including controls that were sacrificed in extremis had alterations among haematology parameters that likely contributed to their demise (e.g. leukemia, neutrophilia, etc); however, the overall number of these cases was low and considered due to naturally-occurring and/or age-related disease that were not related to the test article.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test article-related effects on clinical chemistry parameters up to and including 1000 ppm through 12 months of treatment. At 3500 ppm in males, creatinine was reduced at 6 months, globulin and triglycerides were reduced at 3 months, and albumin/globulin ratio was reduced at 3 months. Urea nitrogen and total bilirubin were slightly increased in the 3500 ppm females at 12 months. However, these differences were minimal, with questionable relation to test article, and were not considered adverse at these magnitudes.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test article-related effects on urinalysis parameters through 12 months of treatment. Specific gravity was increased in 3500 ppm females at 3 months but was considered spurious as it was comparable to control at all other time points.
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
1-year Interim sacrifice

There were no test article-related organ weight changes in interim sacrifice male and female rats. A few statistically significant organ weight changes were observed in interim sacrifice animals (compared to control) but were considered incidental because of their small magnitude, they were secondary to reduced body weight, and/or there was no microscopic correlate and no dose response. In 3500 ppm males, these included increases in relative (to body weight) weights in epididymides, kidneys, and testes (also increased absolute and relative to brain weight). Therefore, they were considered not to be test article related. In 3500 ppm females, these included increases in relative (to body weight) weights in brain and heart.

2-year Terminal sacrifice

There were no test article-related organ weight changes in terminal males or females. Statistically significant organ weight changes (compared to controls) were most likely due to decreased mean body weights or were considered incidental because of their small magnitude and/or the absence of microscopic correlates, and therefore considered not to be test article-related. In 3500 ppm animals, these included increases in relative (to body weight) liver in males and increases in relative (to body weight) brain, heart, liver, and uterus in females; in 3500 ppm females, absolute and relative (to brain) spleen weights were reduced.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
1-year Interim sacrifice

There were no test article-related macroscopic observations in interim sacrifice male and female rats. Macroscopic observations were of the type commonly seen in rats of this age and strain and were considered incidental and unrelated to administration of the test article.

2-year Terminal sacrifice

There were no test article-related macroscopic observations noted in either sex. Macroscopic observations were of the type commonly seen in rats of this age and strain and were considered incidental and unrelated to administration of the test article.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
1-year Interim sacrifice

There were no test article-related microscopic changes in interim sacrifice male and female rats. Focal cortical angiectasis/cystic degeneration was present in the adrenal glands of three interim sacrifice male rats at 3500 ppm but was not observed in control male interim sacrifice rats. This lesion was minimal and unilateral in distribution and was considered to be a spontaneous background observation and not test article related. It occurs more often in females (three control and four 3500 ppm interim sacrifice females) and increases in frequency and severity as rats age. This lesion was not increased in incidence in the main study males or females. Microscopic observations were of the type commonly seen in rats of this age and strain and were considered incidental and unrelated to administration of the test article.

2-year Terminal sacrifice

Test article-related neoplastic and non-neoplastic microscopic observations were noted in testes of male rats. The incidences of interstitial cell hyperplasia and adenoma (benign) in the testes were statistically significant in male rats at 3500 ppm.

A statistically significant trend in testicular interstitial cell adenoma was observed in the 3500 ppm males by the Cochran-Armitage Trend test, but not by Fisher’s Exact test. Four of seven males in the 3500 ppm group with testicular interstitial cell adenomas were those that survived to the termination of the study (Day 730). The remaining three animals were either sacrificed in extremis (Day 679 and Day 721) or were found dead (Day 616) late in the study. As there was a two-fold increase in survival (statistically significant) in the 3500 ppm males, a Peto test was conducted; the resulting p value was 0.009 indicating that the increased incidence in interstitial cell adenoma remained statistically significant when the increase in survival was taken into account.

The test facility historical control range for interstitial adenomas is 0 to 14%. Within the historical control range, there was one study of forty-eight that had a percent incidence (14%) of testicular interstitial cell adenoma greater than that observed in the 3500 ppm males (10%). The remainder of control values were 8.3% or below. The incidence of testicular interstitial cell hyperplasia was also increased in the 3500 ppm males and was statistically significant by both the Fisher’s Exact test and the Cochran-Armitage Trend test. All animals with interstitial cell hyperplasia in the 3500 ppm males were those that survived to the end of the study. However, the percent incidence (11.4%) was outside the laboratory historical control range (0 to 1.5%). Although survival was increased, the majority of adenomas and hyperplasia occurred in terminal or near terminal animals, and the percent incidence of adenomas fell within the historical limits of the laboratory, it is considered likely that the increases in testicular interstitial cell adenoma and hyperplasia in the 3500 ppm males were related to exposure to the test article.

Incidences of testicular interstitial cell adenoma and hyperplasia at 50, 200, and 1000 ppm were well within the test facility historical control ranges and considered unrelated to treatment with the test article.

There were statistically significant increases in two benign tumors in males and two non-neoplastic lesions in females for which the p-value for the Cochran-Armitage Trend Test and/or Fisher’s Exact Test was less than 0.05, which were considered to not be related to treatment.

The Cochran-Armitage Trend Test was significant for benign thyroid follicular cell adenomas in 1000 and 3500 ppm male rats. Fisher’s Exact and Peto Tests were not significant. The incidences of this finding in all male treatment groups were well within the historical control range at the test facility (0 to 11.9%) and did not exhibit a dose related trend. This tumor was not considered treatment related.

The Cochran-Armitage Trend Test was significant for cervical fibromuscular hyperplasia in 3500 ppm females and for cystic endometrial hyperplasia in 200, 1000, and 3500 ppm females. The Fisher’s Exact Test was significant for cystic endometrial hyperplasia in 200 and 1000 Pppm females. The incidences of these findings in all female treatment groups were well within the test facility historical control incidences for cervical fibromuscular and cystic endometrial hyperplasias (0 to 10.0% and 0 to 21.7%, respectively). These non-neoplastic lesions were considered spurious and not related to treatment with the test article.
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
3 500 ppm
System:
male reproductive system
Organ:
testes
Treatment related:
yes
Dose response relationship:
no
Conclusions:
Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for this study was 1000 ppm, equivalent to 45.3 and 57.1 mg/kg/day in males and females, respectively. This NOAEL is based on reduced body weight and nutritional parameters observed in both sexes at 3500 ppm and increased incidence of interstitial cell hyperplasia and benign adenoma in the testes in males at 3500 ppm (equivalent to 162.1 and 203.3 mg/kg/day for males and females, respectively).
Executive summary:

This study was conducted to evaluate the potential chronic toxicity and oncogenicity of the test article when administered over the major portion of the life span of CD® [Crl:CD®(SD)] rats. Five groups of 80 animals/sex/group received untreated diet or the test article orally via dietary admixture approximately 105 weeks (males) or 103 weeks (females) at diet concentrations of 0, 50, 200, 1000, and 3500 ppm. Following 12 months of treatment, ten animals/sex/group were submitted to an interim necropsy.

 

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 weekly. Body weights were measured and recorded weekly from Weeks -1 to 13 and every fourth week thereafter. Food consumption was measured and recorded weekly from Weeks -1 to 13, every fourth week from Weeks 17 to 85, and weekly beginning on Week 88. Body weight gain, food efficiency, and compound consumption were calculated. Ophthalmoscopic examinations were conducted pretest and prior to the interim and terminal necropsy. Blood and urine samples for clinical pathology evaluations were collected from designated animals at 3, 6, and 12 months and from all surviving animals at the terminal necropsy (haematology only). Necropsy examinations were performed, organ weights were recorded, and selected tissues were collected for microscopic examination from 10 animals/sex/group at the interim necropsy on Day 365 and from all remaining animals at the terminal necropsy. Serological health screens were conducted on five unassigned animals/sex pretest and on an additional 15 animals/sex co-housed with study animals at 6, 12, and 18 months.

 

Diets containing 1000 and 3500 ppm were at the targeted concentration and mixed homogeneously throughout the study. Diets containing 50 or 200 ppm were generally at targeted concentrations but were not homogeneously mixed on numerous occasions. This did not impact the study as these groups were not needed to determine the no-observed-adverse- effect level (NOAEL).

 

Over the first year on study, the mean compound consumption in the 50, 200, 1000, and 3500 ppm group was 2.6, 10.4, 52.1, and 185.7 mg/kg/day, respectively, for males and 3.2, 13.0, 64.9, and 228.5 mg/kg/day, respectively, for females. Over the duration of study, the mean compound consumption in the 50, 200, 1000, and 3500 ppm groups was 2.2, 8.8, 45.3, and 162.1 mg/kg/day, respectively, for males and 2.8, 11.0, 57.1, and 203.3 mg/kg/day, respectively, for females.

 

Survival in the 3500 ppm male and female groups and in the 1000 ppm female group was significantly greater than in controls, This increase is likely due to lower body weight in these groups (not statistically significant in females at 1000 ppm). There were no adverse clinical or ophthalmological observations attributed to test article exposure. There was an increase in the incidence of soft faeces at 1000 and 3500 ppm primarily in males that was considered possibly test article-related but not adverse.

 

Mean body weight and body weight gain were reduced during the study in both sexes at 3500 ppm. In males and females, mean body weight was 9% and 17% below control, respectively, at Week 49, and 11% and 17% below control, respectively, on the last weigh day before final sacrifice. Mean body weight gain in this group was 15% and 33% below control for males and females, respectively, over weeks 1 to 49, and 18% and 28% below control, respectively, over the two year exposure period. All of these differences were statistically significant except the male final body weight and overall body weight gain. However, these values were significantly different from control for most of the study. These body weight findings were associated with significantly lower mean food consumption and food efficiency over the first year at this exposure level which continued for the duration of the study (variable statistical significance). Body weight and nutritional parameters in lower concentration groups were generally comparable to control over the study.

 

No test article-related effects were noted on any clinical pathology parameters, organ weights, macroscopic findings, or incidence of masses. There were no test article-related microscopic findings following 1 year of treatment. At the end of the study, statistically significant increases in the incidences of interstitial cell hyperplasia and benign adenoma in the testes were observed in male rats at 3500 ppm. Although survival was increased, the majority of adenomas and hyperplasia occurred in terminal or near terminal animals, and the percent incidence of adenomas fell within the historical limits of the laboratory, it is considered likely that the increases in testicular interstitial cell adenoma and hyperplasia in the 3500 ppm males were related to exposure to the test article.

 

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) was 1000 ppm, equivalent to 45.3 and 57.1 mg/kg/day in males and females, respectively. This NOAEL is based on reduced body weight and nutritional parameters observed in both sexes at 3500 ppm and increased incidence of interstitial cell hyperplasia and benign adenoma in the testes in males at 3500 ppm (equivalent to 162.1 and 203.3 mg/kg/day for males and females, respectively).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
chronic
Species:
rat

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The oncogenic potential of the test substance was assessed in an initial set of chronic feeding studies in rats and mice where the maximum dietary concentration administered was 750 and 800 ppm, respectively. In both studies there was no evidence of a treatment-related effect on the incidence of any specific tumour types. In the 2-year rat study, an apparent slight increase in the incidence of large granular cell leukaemia was noted in males at the highest dose level (750 ppm), which was within the historical control range of the testing facility. This difference from control was concluded to be a reflection of the higher survival rate in the high dose male animals with consequently more animals in this group being at risk for development of this late onset, common spontaneous tumour and, therefore, unrelated to treatment with the test substance.

In a second set of chronic rat and mouse studies, higher dietary concentrations were evaluated (maximums up to 3500 ppm in rats and 4800 ppm in mice). In mice, test article-related and biologically adverse effects were limited to non-neoplastic changes in the duodenum and consisted of increased incidences and severity of mucosal hyperplasia in male mice feed dietary concentrations of 2400 and 4800 ppm. Test article increases in liver weights were observed in males and females fed 2400 and 4800 ppm; the liver weight increase in female mice was associated with microscopic evidence of hepatocellular hypertrophy. Both the liver weight increases and hepatocellular hypertrophy were consistent with hepatic enzyme induction and were interpreted as not adverse. A higher incidence in hepatocellular adenoma (a common spontaneous tumour in mice) in the 4800 ppm male mice was within the range of historical controls from the animal supplier and considered secondary to a marked increase in survival in this group (93%) compared to the concurrent control group (72%). It was concluded that the test substance displays no carcinogenic potential in mice.

The primary observations in the second chronic rat study included a marked increase in survival in males (2-fold relative to controls) and females at 3500 ppm and in females at 1000 ppm, most likely due to the lower body weights in these groups. Reductions in body weight gain, food consumption, and food efficiency were also lower in the high dose group during the first year of the study. At study termination, a statistically significant increase in interstitial (Leydig) cell hyperplasia and benign adenoma in the testes was observed in male rats at 3500 ppm. The incidence of Leydig cell adenoma was within the historical limits of the laboratory and was similar to the upper range of control values reported by the animal supplier. Therefore, the small increase in the incidence of interstitial (Leydig) cell adenoma of the testes observed in high dose males (3500 ppm) in the second 2-year study is insufficient for classification based on the following rationale: 1) Leydig cell adenomas are common in aged rats, and rats are known to be highly sensitive to forming these tumours spontaneously. 2) The maximum tolerated dose was exceeded at the high dose (3500 ppm) where marked reductions in body weight and body weight gain were noted relative to controls. 3) Survival at study termination was markedly increased (2-fold) in the 3500 ppm males (49%) relative to control males (24%), presumably due to the body weight reductions, and thereby put more animals at risk of developing this common spontaneous tumour in aged rats. 4) The incidence of Leydig cell adenoma (10%) was within the range of controls for the testing facility (0-14%) and similar to the maximum for the control range reported by the animal supplier (1.1-9.3%). 5) There was no progression to carcinoma and no decrease in latency period. All tumors were classified as adenoma, and animals bearing Leydig cell adenomas were those that either died late in the study or reached study termination. 6) A very clear threshold was present where the increase in Leydig cell adenoma was observed only at the high dose correlating with the increase in survival at the same dose level. 7) Based on an assessment of the robust genetic toxicity data for this substance, the test substance is not genotoxic. 8) Data mining of the U.S. EPA ToxCast and EDSP21 online databases indicates an overall lack of interaction by the test substance with the estrogen, androgen, and steroidogenesis pathways based on the results of 50 in vitro assays. These data are consistent with the fact that there is no indication within the breadth of toxicology studies conducted with the test substance that it adversely affects any relevant component of the endocrine system that would support a possible relationship of treatment with Leydig cell tumour induction.

Justification for classification or non-classification

Based on the results of chronic feeding studies in rats and mice, 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.