clothianidin (ISO); (E)-1-(2-chloro-1,3-thiazol-5-ylmethyl)-3-methyl-2-nitroguanidine

Carcinogenicity

Description of key information

Key, M-031986-05-1: Combined Chronic Toxicity/Carcinogenicity Studies (52/104 weeks, rat):

NOAEL (chronic toxicity) = 27.4 mg/kg bw/day (male) and 9.7 mg/kg bw/day (female)

LOAEL (chronic toxicity) = 82.0 mg/kg bw/day (male) and 32.5 mg/kg bw/day (female)

NOAEL (carcinogenicity) = ≥ 157 mg/kg bw/day (male) and 193 mg/kg bw/day (female)

Key, M-032363-03-1: Carcinogenicity Studies (78 weeks, mouse):

NOAEL (chronic toxicity) = 47.2 mg/kg bw/day (male) and 65.1 mg/kg bw/day (female)

LOAEL (chronic toxicity) = 171.4 mg/kg bw/day (male) and 215.9 mg/kg bw/day (female)

NOAEL (carcinogenicity) = ≥ 251.9 mg/kg bw/day (male) and 281.1 mg/kg bw/day (female)

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 Oct 1997 - 05 Apr 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

adopted 2018

Deviations:

yes

Remarks:

weight variation at the start of the study was > 20% of mean values, survival rates were less than 50% in 0, 150, 500 and 1500 ppm dose groups (with less than 25% in one female group at 500 ppm), continued under "Principles of methods"

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

adopted 1981

Principles of method if other than guideline:

animals were housed individually, housing temperature slightly different to the guideline

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat was chosen since it is a recommended species for chronic toxicological/cancerogenicity studies in test guidelines.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Inc., North Carolina, USA
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 34 - 40 days
- Weight at study initiation: 105 - 194 g (males) and 113 - 162 g (females)
- Fasting period before study: none
- Housing: individually (except for the first 3 days of acclimation period) in stainless-steel cages, when health problems occurred, some animals were placed in polycarbonate cages
- Diet: certified rodent diet (#8728CM meal), Harlan Teklad, ad libitum
- Water: ad libitum
- Acclimation period: 9 days

DETAILS OF FOOD AND WATER QUALITY:
There were no known contaminants in the diet or water at level that would have interfered with the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 30 - 70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 09 Oct 1997 To: 13 Oct 1999

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): approximately every 2 weeks
- Mixing appropriate amounts with (Type of food): standard diet
- Storage temperature of food: at room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of concentration:
Samples were taken from each dose preparation and stored in a freezer (-10 to -30 °C). Analyses for the concentration of the test material in the dose preparations were done by the testing facility (method MP-TK00-MA). Routine samples from all dose levels beginning with the first mix used for the study and once during month 3, 6, 9, 13, 16, 19, 22, and 25 were analyzed. All samples were stored at room temperature and analyzed within 2 weeks of preparation. The mean concentration of the dose preparation analyses for all levels ranged from 88.7 to 109% of the theoretical concentration.

Homogeneity:
Duplicate samples for homogeneity analyses were taken from the top, middle, and bottom of the dose preparations that bracket the low- and high-dose levels prepared in a prestudy mix. Mean values of the homogeneity analyses ranged from 95.9 to 96.9% and 96.0 to 100% of the theoretical concentration of 100 and 3000 ppm, respectively, thereby indicating a homogenous distribution of the test material.

Stability:
Stability was determined in a separate study at the testing facility by HPLC using a reference standard of the test item. The results of this study evaluation verified that the test article is stable in rodent diet at 25 or 5000 ppm for at least 29 days when stored at room temperature or in a freezer (-10 to -30 °C).

Duration of treatment / exposure:

at least 52 and 104 weeks

Frequency of treatment:

continously via the diet

Dose / conc.:

150 ppm

Remarks:

corresponding to 8.1 mg/kg bw/day (males) and 9.7 mg/kg bw/day (females)

Dose / conc.:

500 ppm

Remarks:

corresponding to 27.4 mg/kg bw/day (males) and 32.5 mg/kg bw/day (females)

Dose / conc.:

1 500 ppm

Remarks:

corresponding to 82.0 mg/kg bw/day (males) and 97.8 mg/kg bw/day (females)

Dose / conc.:

3 000 ppm

Remarks:

corresponding to 157 mg/kg bw/day (males) and 193 mg/kg bw/day (females)

No. of animals per sex per dose:

Terminal sacrifice (104 weeks): 60
Interim sacrifice (52 weeks): 20

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose levels were based on available toxicological data and the results of a previous 13-week toxicity study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once a week

BODY WEIGHT: Yes
- Time schedule for examinations: individual body weights were recorded at randomization, weekly for Week 1 through 14, and over every four weeks thereafter. Terminal body weights were recorded at the scheduled terminal sacrifice. Body weights were also recorded for animals sacrificed at unscheduled intervals.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Individual food consumption data were recorded weekly for Weeks 1 through 13, over four weeks thereafter and at terminal sacrifice.

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

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily during Weeks 12, 25, and 51 for animals designed for sacrafice after 52 weeks of treatment

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Each animal was examined before study initiation and before the respective scheduled sacrifices. The pupils were dilated with 0.5% Mydriacyl and the anterior portion of the eye, optic media, and ocular fundus were examined.
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during Weeks 13, 27, 53, 79, 103/104 (10 females/group) and 105 (10 males/group)
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes, overnight
- How many animals: collected from 10 animals per sex per group per interval (the collections taken during Weeks 13, 27 and 53 were taken from the same animals scheduled for sacrifice after 52 weeks of treatemtn, if possible)
- Parameters checked: haematocrit, haemoglobin concentration, erythrocyte count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, total and differential leukocyte count, platelet count and a measure of blood clotting time/potential, blood cell morphology

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during Weeks 13, 27, 53, 79, 103/104 (10 females/group) and 105 (10 males/group)
- Animals fasted: Yes, overnight
- How many animals: collected from 10 animals per sex per group per interval (the collections taken during Weeks 13, 27 and 53 were taken from the same animals scheduled for sacrifice after 52 weeks of treatemtn, if possible)
- Parameters checked: sodium, potassium, glucose, total cholesterol, blood urea nitrogen, creatinine, total protein and albumin, globulin, albumin/globulin ratio, triglycerides, total bilirubin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyl transpeptidase, creatinine kinase, calcium, inorganic phosphorus, chloride

URINALYSIS: Yes
- Time schedule for collection of urine: approximately 16 hours before blood sampling
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes, overnight
- Parameters checked: appearance, volume, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, blood and sediment

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: functional observation battery (FOB) during Weeks 66 and 67
- Dose groups that were examined: 10 animals per sex per group
- Battery of functions tested: standard cageside and open field measurements and determination of reflexes and physiological parameters

Sacrifice and pathology:

During Week 53 (20 animals per sex and group) and during Weeks 103/104 (females) and 105 (males) animals that were fasted overnight were bled for clinical pathology, anesthetized with sodium pentobarbital, weighted, exsanguinated and necropsied.

GROSS PATHOLOGY: Yes
The necropsy included a macroscopic examination of all orifices, the cranial cavity, the external surface of the brain, the external surface of the spinal cord and cut surfaces of the brain and spinal cord, the cervical tissue and organs, the thoracic, abdominal and pelvic cavities and viscera, the external surface of the body and the nasal cavity and paranasal sinuses.
The following organs were weighted: adrenal, brain, epididymis, heart, kidney, liver, lungs, ovary, pituitary, prostate, spleen, seminal vesicle, testis, thymus, thyroid with parathyroid, uterus with cervix. Organ-to-body weight percentages and organ-to-brain weight ratios were calculated.

HISTOPATHOLOGY: Yes
The following tissues were sampled for histopathology: adrenal, aorta, brain, cecum, cervix, colon, duodenum, epididymis, esophagus, eye, femur with bone marrow, heart, ileum, jejunum, kidney, lesions, liver, lung with mainstem bronchi, lymph node (mandibular and mesenteric), mammary gland (females only), masses and associated tissues, muscle (thigh), optic nerve, ovary, pancreas, pituitary, prostate, rectum, salivary gland, sciatic nerve, seminal vesicle, skin, spinal cord (cervical, thoracic and lumbar), spleen, sternum with bone marrow, stifle joint, stomach, testis, thymus, thyroid with parathyroid, trachea, urinary bladder, uterus, vagina.

Statistics:

One-way analysis of variance was used in order to analyze FOB evaluations, boy weight changes, food consumption, food efficiency, organ weights, organ-to-body weight percentages and organ-to-brain ratios.

Levene's test was done to test for variance homogeneity. In cases of heterogeneity of variance (p<= 0.05), transformations were used. ANOVA was done on the homogeneous or transformed data. If the ANOVA was significant, Dunnett's multiple comparison t-test was used for pairwise comparison between treated and control groups.

Group comparison were evaluated at the 5%, two-tailed probability level.

Incidence of macroscopic and microscopic observations were analyzed for group differences using Fisher-Irwin exact test.

Survival data were analyzed using log-rank test at the 5%, two-tailed probability level for group comparison. The dose-dependency of mortality was tested by the logistic regression method at the 5%, one-tailed probability level.

Each tumor was classified according to the observation described by Peto et al. 1980. When the number of tumor incidences was more than eight, Peto's mortality-prevalence test was used. Otherwise, the exact permutation test was used. Rare tumors were tested at 5% upper-tailed probability level and common tumors were tested at the 1% upper-tailed probability level.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Noted observations were considered to be common findings in CD rats and not related to administration of the test material. For details, please refer to the attached background material (attachment 1).

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

mortality observed, non-treatment-related

Description (incidence):

The overall adjusted survival for rats fed dietary concentration of 0, 150, 500, 1500 and 3000 ppm was 28.0, 29.3, 30.1, 40.0 and 55.0% for males, respectively and 25.3, 26.6, 20.4, 38.5 and 50.4% for females, respectively. Survival to study termination was greater in animals given 1500 and 300 ppm of the test substance compared to the control group. For details, please refer to the attached background material (attachment 2).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related alterations in mean body weights were observed over the course of the study for male rats fed 150 or 500 ppm and for female rats fed 150 ppm. 500 ppm of the test substance led to mildly reduced, but generally statistically significant body weights for females during Week 3 through 13. The lower body weights resulted from reduced food consumption and body weight gain. This effect was considered treatment-related, however, the effect was very mild and the animals recovered body weights over the course of the study.
1500 and 3000 ppm caused a reduced body weight in male and female rats. Over the course of the study, the lower mean body weights ranged from 4.5 to 14% and 9.3 to 21%, respectively for males and females fed with 3000 ppm when compared to the control group. The mean terminal body weights in the high dose group were reduced to 94 and 81% for males and females, respectively. These lower body weights were considered to be an adverse test material-related effect in rats fed with 3000 ppm of the test substance.
Lower body weight gains were also observed for animals in the top dose group. This was also considered to be test material related. The mean overall body weight gains for weeks 1 to 102 for these animals were statistically lowered to 95 and 70% of controls and considered to be an adverse finding due to the magnitude of the reductions. For details, please refer to the attached background material (attachment 3 and 14).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No apparent test material-related effects on food consumption were observed for males fed with 500 ppm and for female rats fed with 150 ppm. However, statistically significantly lower food consumption were sporadically observed for females fed with 150 ppm during Weeks 1 and 6, which were not considered to be treatment-related due to the mild and sporadic nature of the observations. Food consumption was frequently lower for males treated with 1500 and 3000 ppm and for females fed with 500 ppm. These were early effects and may represent a palatability effect. Statistically significantly lower food consumption for females treated with 1500 ppm were observed consistently from Week 1 through 21, with values ranging from 84 to 93% when compared to the control group. From week 25 through 101, the food consumption of those females remained lower, but were not consistently statistically different from the control animals. 3000 ppm of the test substance led to statistically lower food consumption from week 1 to 53 for males and from Week 1 through 77 for females. Thereafter, the food consumption remained lower, although the values were mildly lower and only sporadically significant.
For details, please refer to the attached background material (attachment 4 and 15).

Food efficiency:

no effects observed

Description (incidence and severity):

Statistical analysis was not performed, but the weekly mean food efficiency values measured over the first 13 weeks of the study, indicated that dietary administration at concentrations up to 3000 ppm had no biologically significant effect on the food efficiency. There were no apparent test material-related effects on food efficiency. For details, please refer to the attached background material (attachment 4).

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

There were no obvious test material-related effects on water consumption.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test material-related ophthalmic observations were noted at the Week 53, 103 (females) or 104 (males) examinations. Corneal keratitis and conjunctivitis were both noted at higher incidences for males and females fed 3000 ppm, but are common findings in this age and strain of rat. Therefore, theses finding are not considered to be related to the treatment. For details, please refer to the attached background material (attachment 5).

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Some statistically significant differences in haematological parameters were observed, however, these findings were considered incidental because they were inconsistent over time and exhibited no relationship to dose. For details, please refer to the attached background material (attachment 6 and 16).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

For clinical biochemistry some statistically significant or otherwise notable differences were observed. Most of the differences were considered incidental because they were inconsistent over time and exhibited no relationship to dose. The only consistent difference was the mildly higher inorganic phosphorus for males fed with 3000 ppm which was considered due to the test material administration. Although this difference was minor, it was statistically significant at most test intervals. This effect may be related to the histopathological finding of increased incidence and severity of renal pelvis mineralization. For details, please refer to the attached background material (attachment 7 and 16).

Endocrine findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Some statistically significant differences in urinalysis data were observed, however, these findings were considered incidental because they were inconsistent over time and exhibited no relationship to dose. For details, please refer to the attached background material (attachment 8).

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There were no obvious test material-related effects on FOB evaluations. Noted observations were considered to be common findings in CD rats and not related to administration of the test material. Forelimb grip strength was statistically higher for females given 150 or 3000 ppm. This effect was not dose-dependent and not observed for males. Therefore, this effect was not considered to be treatment-related. For details, please refer to the attached background material (attachment 9).

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Interim sacrifice:
Organ-to-body weight percentage of adrenal gland, brain, heart, kidney, liver, lung, spleen and uterus were significantly greater in female rats fed with 3000 ppm when compared to the control animals. The absolute weights of these organs did not differ from the controls and no microscopic findings were observed. Therefore, these differences were considered secondary to the lower body weights of these animals. However, increased relative liver weights in females may have correlated with histipathological findings. The liver-to-body weight percentage in the females fed with 150 or 1500 ppm and the liver-to-brain weight percentage in the females given 150 ppm were also slightly greater compared to the control animals.
In males, the organ-to-body weight percentage for brain, liver and lung were significantly greater in the high dose group compared to the control group. However, the absolute organ weights were not altered compared to the control animals and no microscopic changes were observed, these effects were not considered to be toxicologically significant.

Terminal sacrifice:
The organ-to-body weight percentage of female brain, heart, kidney, liver, and lung were significantly greater in the high dose group than in the control group. However, the absolute organ weights did not differ from controls and no microscopic changes were observed. Thus, these effects were considered to be secondary to the lower body weights in these animals. However, increased relative liver weights may have correlated with histipathological findings. The thymus-to-body weight percentage in females fed with 150 ppm was significantly lower than the controls. However, there were no differences in the absolute thymus weight compared to the control animals and no microscopic findings. Furthermore, the mean terminal body weight of these female rats were the highest, whereas the mean thymus weight of this group was the lowest. Therefore, the significantly lower thymus-to-body weight percentage was considered an incidental occurrence secondary.
No significant differences in organ weights were observed between control and treated male rats.

For details, please refer to the attached background material (attachment 10 and 17).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Several macroscopic findings were observed between the treated and the control animals. However, many of these effects were considered not to be related to the administration of the test substance because they were not observed in a dose related-manner, were considered to be related to increased survival in the treated groups, or both.

Effects that were considered related to the administration of the test substance, or had an unclear relationship to treatment included effects in the glandular stomach, liver and kidney.

Glandular stomach:
In the high dose group, incidences of macroscopically observed dark focus/area in the glandular stomach were increased. These correlated with significantly increased incidences of hemorrhage in the glandular stomach in males fed with 3000 ppm and of erosion of the glandular stomach mucosa in females given 3000 ppm. Further, incidences of edema of the granular stomach wall were significantly increased in male and female rats fed with 3000 ppm of the test material. It was considered that these were indicative of irritant effects.

Kidney:
Granular material or calculus were observed in kidneys of a few male rats given 3000 ppm and correlated microscopically with an increased amount of pelvic mineralization in that group.

Liver:
Mottled livers were seen in males of the 1500 and 3000 ppm groups.

For details, please refer to the attached background material (attachment 11).

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Several differences were observed in microscopic findings between the treated and the control animals. However, many of these effects were considered not to be related to the administration of the test substance because they were not observed in a dose related-manner, were considered to be related to increased survival in the treated groups, or both.
Effects that had an unclear relationship to treatment included effects in the kidney (please refer to gross pathological findings), liver and in the ovary:
Instances of liver eosinophilic foci were increased in animals in the top dose group after terminal sacrifice.
Microscopically, incidences of hyperplasia of the interstitial glands of the ovary were significantly increased in female rats fed with 500, 1500 or 3000 ppm of the test substance when compared to the control animals. Interstitial gland hyperplasia was seen in ovaries undergoing atrophy with little or no active follicular development, and appeared to be an aging change. Furthermore, the incidence of interstitial gland hyperplasia for all study groups were within the range of control female rats (same strain and study duration) at the same testing facility (historical control data from 1994 to 2002). The toxicological significance of the interstitial gland hyperplasia was evaluated in an additional statement (2015, M-524671-01-1). In this statement, the ovarian lesions were considered not to be toxicologically significant due to several reasons:
1) The proliferation of stromal (interstitial) cells in the ovaries of rodents does not have a counterpart in the ovaries of human adult females and is not considered relevant for human safety assessment.
2) The incidences of interstitial gland hyperplasia were within the testing laboratory's historical control data range.
3) There is no evidence that the test substance directly affects reproductive or endocrine organs not only in the rat carcinogenicity study but in the other studies.4) It is likely that severe suppression of body weight contributes to an increased incidence of interstitial gland hyperplasia.
5) The test substance did not affect the reproductive organs directly or have an endocrine disrupting function. It is considered likely that this finding was secondary as the result of treatment with excessive doses causing marked to severe suppression of body weight and associated stress, rather than any direct effect. Therefore, it is concluded that the increased incidence of interstitial gland hyperplasia observed in the treated animals is not of toxicological significance.

For details on histopathological findings, please refer to the attached background material (attachment 12 and 18).

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no neoplastic lesions that were considered to be related to administration of the test material.

Three hepatocellular carcinomas were present in males fed with 500 ppm (1 at terminal sacrifice and 2 in unscheduled deaths) and in males at 3000 ppm (4 in unscheduled deaths). The incidence of hepatocellular carcinoma for males fed 3000 ppm was statistically increased. The tumors were considered unlikely to be treatment-related owing to the absence of any dose-effect relationship and the low incidence (exceeding only slightly laboratory historical control data). Incidences and average severities of eosinophilic altered hepatocellular focus were significantly increased in rats fed with 3000 ppm of the test material. The eosinophilic foci and the hepatocellular neoplasms differed significantly in microscopic appearance and there appeared to be no relationship between the eosinophilic foci and the hepatocellular carcinomas.
In male rats, there was a significant increase of thyroid C-cell adenoma incidences in the mid- and mid-high dose group but not in the high dose group compared to the control. This was not associated with any monotonic dose-response. Therefore, these incidences are not considered treatment-related.

In female rats there were several inconsistent increases of neoplastic lesions. They included the thyroid C-cell adenoma and carcinoma, the adrenal medulla-benign and malignant pheochromocytoma, the mammary carcinoma, adenoma and fibroadenoma. All of the above increases were not associated with significant trends and are, therefore, considered to be biologically irrelevant.
In regards to the C-cell tumors observed in female rats, an independent review of the original histopathology of the female rat thyroid was undertaken by a consensus diagnosis for each animal. This consensus pathology review confirmed that the observed thyroid C-cell adenomas were not treatment-related. Furthermore, it was concluded, that there was no evidence of progression of the thyroid C-cell hyperplasia to adenoma noted in the study.

Lower incidences of neoplasia were observed in males and females for e.g. thyroid follicular cell adenoma, kidney-tubular cell adenoma, pituitary adenoma and carcinomas and pancreatic islet cell adenoma and carcinoma.
For details, please refer to the attached background material (attachment 13 and 18).

Other effects:

not examined

Description (incidence and severity):

not applicable

Key result

Dose descriptor:

NOAEL

Remarks:

chronic toxicity

Effect level:

150 ppm (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: corresponding to 9.7 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

chronic toxicity

Effect level:

500 ppm (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no aderse effects observed at this dose level

Remarks on result:

other: corresponding to 27.4 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

3 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment-related neoplastic findings observed at this dose level

Remarks on result:

other: corresponding to 157 mg/kg bw/day (males) and 193 mg/kg bw/day (females)

Key result

Dose descriptor:

LOAEL

Remarks:

chronic toxicity

Effect level:

500 ppm (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

histopathology: non-neoplastic

Remarks on result:

other: corresponding to 32.5 mg/kg bw/day (females)

Key result

Dose descriptor:

LOAEL

Remarks:

chronic toxicity

Effect level:

1 500 ppm (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

histopathology: non-neoplastic

Remarks on result:

other: corresponding to 82.0 mg/kg bw/day

Key result

Critical effects observed:

no

Conclusions:

The study was performed under GLP conditions and according to OECD TG 453 (adopted 1981). Deviations to the current version (adopted 2018) are minor. Thus the studies are considered reliable and valid. Based on nature, incidence or chronology of neoplasms, or from the numbers of animals exhibiting tumors in treated and control rats, there was no indication of a treatment-related oncogenic effect of the test item. Under the conditions of this study, the NOAEL for chronic toxicity was considered to be 500 ppm (27.4 mg/kg bw/day) for males and 150 ppm (9.7 mg/kg bw/day) for females based on treatment related reductions in body weights and food consumption in males fed 1500 ppm and above and in females fed 500 ppm and above, and histopathology findings in males fed 3000 ppm and females fed 500 ppm and above.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 Oct 1997 - 20 Mar 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

adopted 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

adopted 1981

GLP compliance:

yes

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Inc., Michigan, USA
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 35 - 41 days
- Weight at study initiation: 19.8 - 33.8 g (males) and 15.8 - 25.9 g (females)
- Fasting period before study: none
- Housing: individually (except for the first 7 days of acclimation period) in stainless-steel cages; when health problems occurred, some animals were placed in polycarbonate cages
- Diet: Rodent diet (#8728CM meal), Harlan Teklad, ad libitum
- Water: ad libitum
- Acclimation period: 11 days

DETAILS OF FOOD AND WATER QUALITY:
There were no known contaminants in the diet or water at level that would have interfered with the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 30 - 70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 10 Oct 1997 To: 19 Apr 1999

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): approximately every 2 weeks
- Mixing appropriate amounts with (Type of food): standard diet
- Storage temperature of food: room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of concentration:
Samples were taken from each dose preparation and stored in a freezer (-10 to -30 °C). Analyses
for the concentration of the test material in the dose preparations were done by the testing facility
(method MP-TK00-MA). Routine samples from all dose levels beginning with the first mix used for the study and once during Month 3, 6, 9, 12, 15 and 18 were analyzed. All samples were stored at room temperature and analyzed within 2 weeks of preparation. With the exception of the 100 and 350 ppm samples from Week 61/62 preparation, the mean concentration of the dose preparation analyses for all levels ranged from 86.0 to 107% of the theoretical concentration. When samples from the Week 61/62 preparation were retested, the mean results were within acceptable limits.

Homogeneity:
Duplicate samples for homogeneity analyses were taken from the top, middle, and bottom of the dose preparations that bracket the low- and high-dose levels prepared in a prestudy mix. Mean values of the homogeneity analyses ranged from 93.4 to 97.2% and 97.2 to 99.6% of the theoretical concentration of 50, 1500 and 2500 ppm, respectively.

Stability:
Stability was determined in a separate study at the testing facility by HPLC using a reference standard of the test item. The results of this study evaluation verified that the test article is stable in rodent diet at 25 or 5000 ppm for at least 29 days when stored at room temperature or in a freezer (-10 to -30°C).

Duration of treatment / exposure:

78 weeks

Frequency of treatment:

continously via the diet

Dose / conc.:

100 ppm

Remarks:

corresponding to 13.5 mg/kg bw/day (males) and 17.0 mg/kg bw/day (females)

Dose / conc.:

350 ppm

Remarks:

corresponding to 47.2 mg/kg bw/day (males) and 65.1 mg/kg bw/day (females)

Dose / conc.:

1 250 ppm

Remarks:

corresponding to 171.4 mg/kg bw/day (males) and 215.9 mg/kg bw/day (females)

Dose / conc.:

2 000 ppm

Remarks:

Animals received:
700 ppm for Weeks 1 to 4;
2000 ppm for Weeks 5 to 10;
2500 ppm for Weeks 11 to 34;
2000 ppm for males and 1800 ppm for females from Week 35 to termination;
corresponding to 251.9 mg/kg bw/day (males) and 281.1 mg/kg bw/day (females)

No. of animals per sex per dose:

50

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
Dose levels were based on available toxicological data and the results of a previous 13-week toxicity study. During the first 4 weeks of the study minimal signs of toxicity were observed in animals fed 1250 ppm (the initial high dose). Therefore the dietary concentration to the 700 ppm dose group was raised at Week 5 to 2000 ppm. At this dose level it was not apparent that a maximum tolerated dose (MTD) would be achieved. Therefore the dietary concentration was increased to 2500 ppm which resulted in substantial body weight and mortality effects. Based on the noted observations, the dietary concentration of the high dose group was lowered at Week 35 to 2000 ppm for males and 1800 ppm for females.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once a week

BODY WEIGHT: Yes
- Time schedule for examinations: individual body weights were recorded one the first day of treatment, weekly for Week 1 through 14, and over every four weeks thereafter and Week 55. Body weights were also recorded for animals sacrificed at an unscheduled interval.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Individual food consumption data were recorded weekly for Weeks 1 through 14, and every fourth week thereafter.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during Week 53 and at the scheduled sacrifice, blood from animals sacrificed at an unscheduled interval was also collected
- Anaesthetic used for blood collection: Yes (carbon dioxide/oxygen inhalation)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: differential blood cell count (nucleated red blood cells, segmented neutrophils, band neutrophils, lmyphocytes, monocytes, eosinophils, basophils); differential leukocyte counts and cellular morphology quantitations were manually determined from peripheral blood smears from all animals sacrificed at an unscheduled interval and from control and high dose animals after 52 and 78 weeks of treatment

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

A necropsy was conducted on each animal that died or was sacrificed at an unscheduled interval. Animals were sacrificed by anaesthesia with sodium pentobarbitol followed by exsanguination. During weeks 79 and 80, animals were fasted for at least 4 hours prior to sacrifice and necropsy.

GROSS PATHOLOGY: Yes
The necropsy included a macroscopic examination of all orifices, the cranial cavity, the external surface of the brain, the external surface of the spinal cord and cut surfaces of the brain and spinal cord, the cervical tissue and organs, the thoracic, abdominal and pelvic cavities and viscera, the external surface of the body and the nasal cavity and paranasal sinuses.
The following organs were weighted: adrenal, brain, epididymis, heart, kidney, liver with drained gallbladder, lungs, ovary, spleen and testes.
Organ-to-body weight percentages and organ-to-brain weight ratios were calculated.

HISTOPATHOLOGY: Yes
The following tissues were preserved in 10% buffered formalin for subsequent histopathological examination: gross lesions, brain, spinal cord (at three levels: cervical, mid-thoracic and lumbar), salivary glands, harderian gland, oesophagus, stomach, small and large intestines, liver, pancreas, kidneys, adrenals, spleen, head, heart, trachea and lungs, larynx, aorta, gonads, uterus, accessory sex organs, female mammary gland, prostate, urinary bladder, lymph nodes, femur with bone marrow, sternum with bone marrow, muscle, thyroid and parathyroid, optic nerve, sciatic nerve, thymus, skin and eyes.

Statistics:

One-way analysis of variance (ANOVA, Winer, 1971) was used to analyse body weights, body weight changes, food consumption, food efficiency, organ weights, organ-to-body weight percentages and organ-to-brain weight ratios.

Levene’s test was done to test for variance homogeneity. In the case of heterogeneity of variance at p≤ 0.05, transformations were used to stabilise the variance (Conover et al., 1981; Dixon et al, 1969). ANOVA was done on the homogeneous or transformed data. If ANOVA was significant, Dunett’s multiple comparison t-test was used for pairwise comparisons between treated and control groups.

Group comparisons were evaluated at the 5.0% two-tailed probability level.

Incidence of macroscopic and microscopic observations were analysed for group differences using the Fisher-Irwin exact test.

Survival data were analysed using the log-rank test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Dose level dependent non-traumatic vocalisation was observed predominantly in males and females in the top two dose groups. This observation was first noted during Week 7 for animals in the mid-high and high-dose groups and continued to be noted until Week 80. For details on the observed clinical signs, please refer to the attached background material (attachment 1).

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Survival for males was 68, 84, 60, 80 and 68% in groups fed with diet only, 100, 350, 1250 and 2000 ppm, respectively. There was a statistically significant increased mortality for females given 350 or 1800 ppm, however this was not dose dependent. Survival for female mice given 0, 100, 350, 1250, 1800 ppm was 80, 86, 58, 76 and 46%, respectively. For details, please refer to the attached background material (attachment 2).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Treatment related statistically significant decreases in mean body weights were observed for most animals of the study in the top dose group and for much of the study in the 1250 ppm dose group. These decreased body weights were observed during weeks 6 through 78 and weeks 10 through 78 for high-dose males and females, respectively. When compared to the control animals, the decrease in mean body weights of the high-dose males ranged from 82.2 to 95.8 % and of the high-dose females from 82.9 - 95.3%.
The lower body weights in the top dose group generally correlated with lower mean food consumption. Treatment related statistically significant decreases in mean body weight gains were measured for females in the top two dose groups and males in the top dose group. For high dose animals body weight decrements occurred in a dose dependent manner as the dietary concentration for this group was altered. For details, please refer to the attached background material (attachment 3 and 10).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption values were statistically significantly lower throughout much of the study for the top dose group (79.0 - 92.5% for males and 75.9 - 88.9% for females) when compared to the control group. The lower mean food consumption values generally corresponded to lower mean body weights of the high-dose group animals. For the animals of the mid-high dose dose group, the mean food consumption values were similar to those of the control animals. A few incidences of statistical significant differences (higher and lower) in mean food consumption values were noted for animals fed 100 or 350 ppm when compared to the respective control group. However, these alterations were transient, small in magnitude and not dose-related.
For details, please refer to the attached background material (attachment 4 and 11).

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

The overall mean food efficiency values indicate that dietary adminsitration at concentration up to 1250 ppm had no biologically significant effect on this parameter. For animals in the high-dose group, the decrements in food efficiency occurred in a dose-related manner as the dietary concentration for this group was altered, similar to what was observed for body weight gains. For details, please refer to the attached background material.

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically significant treatment related findings for the relative (%) differential white blood cell counts. Statistically significant differences for differential cell counts were considered unrelated to treatment as they were small, inconsistent over time and between the sexes. A slight increase in the neutrophil fraction (and a concomitant decrease in lymphocyte fraction) was observed in top dose females at terminal sacrifice which may be correlated with the stressful and underfeeding status of this group. For further details, please refer to the attached background material (attachment 5 and 12).

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not applicable

Endocrine findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Absolute brain, kidney, liver/gallbladder weights were significantly reduced in females given 1800 ppm and the trend for decreased liver/gallbladder weight extended to females given 1250 ppm. Absolute and relative ovary weights were significantly increased in females given 1800 ppm. Significant organ weight reductions were also recorded in the heart and kidneys of males given 350, 1250 or 2000 ppm and this trend extended to the kidneys of males given 100 ppm. Specific correlates for most organ weight changes were not observed microscopically. The minor alterations in these organ weights are common findings in this strain of mouse and were considered not to be treatment-related or toxicologically significant. In most cases there was an effect on relative organ weight. The reduced absolute liver, heart and kidney weights were attributed to degenerative changes associated with systemic amyloidosis. The organ weight alterations observed at the higher dose levels may have been, in part, secondary to observed body weight decreases. For details, please refer to the attached background material (attachment 6 and 13).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A broad spectrum of macroscopic changes was recorded in both sexes at terminal sacrifice. However, no macroscopic observations were directly attributed to ingestion of the test substance.For details, please refer to the attached background material (attachment 7).

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment related microscopic changes in animals that died at an unscheduled interval or were sacrificed terminally were restricted to the liver of both sexes and generally consisted of minimal to moderate centrilobular to panlobular hepatocellular hypertrophy. In females this change was largely restricted to animals fed 1250 or 1800 ppm, however in males increased liver cell hypertrophy was noted in animals fed 100, 1250 or 2000 ppm. Although there was no apparent dose response this is considered to be a test material effect which is a common pharmacological response to a xenobiotic and considered not adverse. Slightly increased fibromuscular hyperplasia of the cervix in females at 1250 and 1800 ppm may have been treatment related. Increased myocardial degeneration in males at 1250 ppm did not show a dose response relationship and was unlikely to be treatment related. For details, please refer to the attached background material (attachment 8 and 14).

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Neoplastic diseases observed in this study were generally consistent with common, spontaneous tumors in CD-1 mice. No tumor variants were increased in a dose-related manner. Microscopic findings indicate that the test material was not carcinogenic for male and female mice during this study. For details, please refer to the attached background material (attachment 9).

Key result

Dose descriptor:

NOAEL

Remarks:

chronic toxicity

Effect level:

350 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: corresponding to 47.2 mg/kg bw/day in males and 65.1 mg/kg bw/day in females

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

2 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment-related neoplastic findings observed at this dose level

Remarks on result:

other: corresponding to 251.9 mg/kg bw/day (males) and 281.1 mg/kg bw/day (females)

Key result

Dose descriptor:

LOAEL

Remarks:

chronic toxicity

Effect level:

1 250 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: corresponding to 171.4 mg/kg bw/day (males) and 215.9 mg/kg bw/day (females)

Key result

Critical effects observed:

no

Conclusions:

The study was performed under GLP conditions and according to OECD TG 451 (adopted 1981). Deviations to the current version (adopted 2018) are minor. Thus, the study is considered reliable and valid. Based on nature, incidence or chronology of neoplasms, or from the numbers of animals exhibiting tumors in treated and control mice, there was no indication of a treatment-related oncogenic effect of the test item. Under the conditions of this study, the NOAEL for chronic toxicity was considered to be 350 ppm for male and female mice which corresponds to 47.2 mg/kg bw/day for males and 65.1 mg/kg bw/day for females. This value is based on treatment-related reductions in body weights, body weight gain and food consumption.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 1) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex X, 8.9.1, of Regulation (EC) No 1907/2006.

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

Please refer to the information provided in the endpoint summary.

Justification for classification or non-classification

The available data on the carcinogenicity of the substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.

Additional information

The chronic and carcinogenic effects of the substance were investigated in male and female Wistar (Crl:CD(SD)IGS BR) rats in a combined study of chronic toxicity and carcinogenicity for 104 weeks according to OECD guideline 453 and GLP (M-031986-05-1, 2000). Eighty animals per sex and group were exposed to the test substance at dietary levels of 150, 500, 1500, and 3000 ppm during the chronic (20 animals per sex and dose) and carcinogenic (60 animals per sex and dose) exposure. For the carcinogenicity study, the dietary dose levels corresponded to a mean achieved compound intake of 8.1, 27.4, 82.0 and 157 mg/kg bw/day in males and 9.7, 32.5, 97.8, 193 mg/kg bw/day in females, respectively (as calculated from the reported body weight and food intake values during Weeks 1-101).

The overall adjusted survival for rats at 0, 150, 500, 1500 or 3000 ppm was 28.0, 29.3, 30.1, 40.0 and 55.0 % for males, respectively and 25.3, 26.6, 20.4, 38.5 and 50.4% for females, respectively. With exception of one dose group (500 ppm, females) the adjusted survivals were above 25% as recommended by the OECD guideline. It should be noted, that the terminal survival of the female rats given 500 ppm was below 25%, however, the survival rates in total were considered to be scientifically acceptable.

There were no obvious test material-related effects on clinical sings, ophthalmic observations or on the functional observational battery (FOB).

In regards to body weight, no treatment-related alterations were observed over the course of the study for male rats fed 150 or 500 ppm and for female rats fed 150 ppm. 500 ppm of the test substance led to mildly reduced, but generally statistically significant body weights of females during Week 3 through 13. The lower body weights resulted from reduced food consumption and body weight gain. This effect was considered treatment-related, however, the effect was very mild and the animals recovered body weights over the course of the study. 1500 and 3000 ppm caused a reduced body weight in male and female rats. Over the course of the study, the lower mean body weights ranged from 5 to 14% and 9 to 21%, respectively for males and females fed with 3000 ppm when compared to the control group. The mean terminal body weights in the high dose group were reduced to 94 and 81% for males and females, respectively. These lower body weights were considered as adverse in rats fed with 3000 ppm of the test substance. Lower body weight gains were also observed for animals in the top dose group. This was also considered to be test material-related. The mean overall body weight gains for weeks 1 to 102 for these animals were statistically lowered to 95 and 70% of controls.

Food consumption was frequently lower for males treated with 1500 and 3000 ppm and for females fed with 500 ppm. These were early effects and may represent a palatability effect. Statistically significantly lower food consumption for females treated with 1500 ppm were observed consistently from Week 1 through 21, with values ranging from 84 to 93% when compared to the control group. From week 25 through 101, the food consumption of those females remained lower, but was not consistently statistically different from the control animals. 3000 ppm of the test substance led to statistically lower food consumption from Week 1 to 53 for males and from Week 1 through 77 for females. Thereafter, food consumption remained mildly lower, although only sporadically significant. There were no apparent test substance-related effects on food efficiency or water consumption.

Some statistically significant differences in hematological parameters and clinical biochemistry were observed, however, these findings were considered incidental because they were inconsistent over time and exhibited no relationship to dose. The only consistent difference was the mildly higher inorganic phosphorus level in males fed with 3000 ppm, which was considered due to the test material administration. Although this difference was minor, it was statistically significant at most test intervals. This effect may be related to the histopathological finding of increased incidence and severity of renal pelvis mineralization.

The organ-to-body weight percentage of a number of organs in females fed 3000 ppm were significantly increased when compared to the control animals. However, these differences were considered secondary due to the lower body weights of females at this dose.

Several differences were observed between the treated and the control animals in gross and histopathology. However, many of these effects were considered not to be related to the administration of the test substance because they were not observed in a dose-related manner, were considered to be related to increased survival in the treated groups, or both. Effects that were considered related to the administration of the test substance, or had an unclear relationship to treatment included effects in the glandular stomach, liver, kidney and ovary.

Glandular stomach: In the high dose group, incidences of dark focus/area in the glandular stomach were observed. These correlated with significantly increased incidences of hemorrhage in the glandular stomach in males fed with 3000 ppm and of erosion of the glandular stomach mucosa in females given 3000 ppm. Further, incidences of edema of the granular stomach wall were significantly increased in male and female rats fed with 3000 ppm of the test material. The significance of these findings is unclear but they might indicate local effects due to irritation.

Liver: Mottled livers were seen in males of the 1500 and 3000 ppm groups. Further, instances of liver eosinophilic foci were increased in animals in the top dose group after terminal sacrifice.

Kidney: Granular material (4/60 males) or calculus (2/60 males) were observed in kidneys of rats given 3000 ppm and correlated microscopically with an increased amount of pelvic mineralization in that group.

Ovary: Microscopically, incidences of hyperplasia of the interstitial glands of the ovary were significantly increased in female rats fed with 500, 1500 or 3000 ppm of the test substance when compared to the control animals. Interstitial gland hyperplasia was seen in ovaries undergoing atrophy with little or no active follicular development, and appeared to be an aging change. Furthermore, the incidence of interstitial gland hyperplasia for all study groups were within the range of control female rats (same strain and study duration) at the same testing facility (historical control data from 1994 to 2002). The toxicological significance of the interstitial gland hyperplasia was evaluated in an additional statement (M-524671-01-1, 2015). In this statement, the ovarian lesions were considered not to be toxicologically significant due to several reasons:

1) The proliferation of stromal (interstitial) cells in the ovaries of rodents does not have a counterpart in the ovaries of human adult females and is not considered relevant for human safety assessment.

2) The incidences of interstitial gland hyperplasia were within the testing laboratory's historical control data range.

3) There is no evidence that the test substance directly affects reproductive or endocrine organs not only in the rat carcinogenicity study but in the other studies. Therefore, it is considered that the test substance does not exacerbate the incidence of "interstitial gland hyperplasia" by a direct endocrine disrupting effect via stimulation of the hypothalamic-pituitary-gonadal axis.

4) It is likely that severe suppression of body weight contributes to an increased incidence of interstitial gland hyperplasia.

5) The test substance did not directly affect the reproductive organs or have an endocrine disrupting function. It is considered likely that this finding was secondary as the result of treatment with excessive doses causing marked to severe suppression of body weight and associated stress, rather than any direct effect. Therefore, it is concluded that the increased incidence of interstitial gland hyperplasia observed in the treated animals is not of toxicological significance.

There were no neoplastic lesions that were considered to be related to administration of the test material. However, incidences of neoplasms occurred as follows:

Hepatocellular carcinomas: The incidence of hepatocellular carcinoma for males fed 3000 ppm was statistically increased. The tumors were considered unlikely to be treatment-related owing to the absence of any dose-effect relationship and the low incidence (exceeding only slightly laboratory historical control data). Incidences and average severities of eosinophilic altered hepatocellular foci were significantly increased in rats fed with 3000 ppm of the test material. The eosinophilic foci and the hepatocellular neoplasms differed significantly in microscopic appearance and no relationship between the eosinophilic foci and the hepatocellular carcinomas was evident.

Thyroid C-cell adenomas: In male rats, there was a significant increase of thyroid C-cell adenoma incidences in the mid- and mid-high dose group compared to the control, but not in top dose males. Thus, no association to any monotonic dose-response was apparent. Therefore, these incidences are not considered treatment-related. In female rats, there were several inconsistent increases of neoplastic lesions. They included the thyroid C-cell adenoma and carcinoma, the adrenal medulla-benign and malignant pheochromocytoma, the mammary carcinoma, adenoma and fibroadenoma. All of the above-mentioned increases were not associated with significant trends and are, therefore, considered to be biologically irrelevant. In regards to the C-cell tumors observed in female rats, an independent review of the original histopathology of the female rat thyroid was undertaken by a consensus diagnosis for each animal. This consensus pathology review confirmed that the observed thyroid C-cell adenomas were not treatment-related. Furthermore, it was concluded, that there was no evidence of progression of the thyroid C-cell hyperplasia to adenoma noted in the study.

Overall, the combined chronic toxicity/carcinogenicity study was performed under GLP conditions and according to OECD TG 453 (adopted 1981). Deviations to the current version (adopted 2018) are minor. Thus, the study is considered reliable and valid. Based on nature, incidence or chronology of neoplasms, or from the numbers of animals exhibiting tumors in treated and control rats, there was no indication of a treatment-related oncogenic effect of the test item. Under the conditions of this study, the NOAEL for chronic toxicity was considered to be 500 ppm (27.4 mg/kg bw/day) for males and 150 ppm (9.7 mg/kg bw/day) for females based on treatment-related reductions in body weights and food consumption in males fed 1500 ppm and above and in females fed 500 ppm and above, and histopathology findings in males fed 3000 ppm and females fed 500 ppm and above.

 

In a further carcinogenicity study performed according to OECD 451 and GLP, male and female mice (Crl:CD-1(ICR)BR VAF/Plus) were assigned to five groups (50/sex/group). Each group received diets containing basal diet or test substance at 100, 350, 1250 and 1800 (females) or 2000 ppm (males) for 78 weeks, which corresponded to 13.5, 47.2, 171.4 and 251.9 mg/kg bw/day for males and 17.0, 65.1, 215.9, 281.1 mg/kg bw/day for females (M-032363-03-1, 2000).

Survival for males was 68, 84, 60, 80 and 68% in groups fed with diet only, 100, 350, 1250 and 2000 ppm, respectively. There was a statistically significant increased mortality in females given 350 or 1800 ppm, however, this was not dose-dependent. Survival of female mice given 0, 100, 350, 1250, 1800 ppm was 80, 86, 58, 76 and 46%, respectively.

Dose level-dependent non-traumatic vocalization was observed predominantly in males and females in the top two dose groups. This was noted predominantly for animals in the mid-high and high dose groups, starting during Week 7 and continuing to be noted to Week 80. It was noted in the other dose groups only during Week 69.

Treatment-related statistically significant decreases in mean body weights were observed in the mid-high and high dose groups. When compared to the control animals, the decrease in mean body weights of the high-dose males ranged from 82.2 to 95.8% and of the high-dose females from 82.9 - 95.3%. The decreases in body weight and body weight gains indicated that the maximum tolerated dose (MTD) was exceeded at the high dose. Males in the mid-high dose group also had reduced body weights at intervals during the study, this was also considered to be test material-related. The lower body weights in the top dose group generally correlated with lower mean food consumption. Treatment-related statistically significant decreases in mean body weight gains were measured for females in the top two dose groups and males in the top dose group. For high dose animals body weight decrements occurred in a dose-dependent manner as the dietary concentration for this group was altered. For animals in the high-dose group, the decrements in food efficiency occurred in a dose-related manner as the dietary concentration for this group was altered, similar to what was observed for body weight gains. There were a few incidences in which the mean food consumption values of animals in the low and mid-dose groups were significantly different (lower and higher) than control. The differences were small in magnitude, transient and not dose-related, therefore were considered spurious.

There were no toxicologically significant treatment-related findings for the relative (%) differential white blood cell counts. Statistically significant differences were considered unrelated to treatment as they were small, inconsistent over time and between the sexes.

Absolute brain, kidney, liver/gallbladder weights were significantly reduced in females given 1800 ppm and the trend for decreased liver/gallbladder weight extended to females given 1250 ppm. Absolute and relative ovary weights were significantly increased in females given 1800 ppm. Significant organ weight reductions were also recorded in the heart and kidneys of males given 350, 1250 or 2000 ppm and this trend extended to the kidneys of males given 100 ppm. Specific correlates for most organ weight changes were not observed microscopically. The minor alterations in these organ weights are common findings in this strain of mouse and were considered not to be treatment-related or toxicologically significant. In most cases, there was an effect on relative organ weight. The reduced absolute liver, heart and kidney weights were attributed to degenerative changes associated with systemic amyloidosis. The organ weight alterations observed at the higher dose levels may have been, in part, secondary to observed body weight decreases.

A broad spectrum of macroscopic changes was recorded in both sexes at terminal sacrifice. However, no macroscopic observations were directly attributed to ingestion of the test substance.

Treatment-related microscopic changes in animals that died at an unscheduled interval or were sacrificed terminally were restricted to the liver of both sexes and generally consisted of minimal to moderate centrilobular to panlobular hepatocellular hypertrophy. In females, this change was largely restricted to animals fed 1250 or 1800 ppm, however, in males increased liver cell hypertrophy was noted in animals fed 100, 1250 or 2000 ppm. Although there was no apparent dose-response this is considered to be a test material effect which is a common pharmacological response to a xenobiotic and considered not adverse.

Neoplastic lesions observed in this study were generally consistent with common, spontaneous tumors in CD-1 mice. No tumor variants were increased in a dose-related manner. Microscopic findings indicate that the test material was not carcinogenic for male and female mice during this study.

Overall, the study was performed under GLP conditions and according to OECD TG 451 (adopted 1981). Deviations to the current version (adopted 2018) are minor. Thus, the study is considered reliable and valid. Based on nature, incidence or chronology of neoplasms or from the numbers of animals exhibiting tumors in treated and control mice, there was no indication of a treatment-related oncogenic effect of the test item. Under the conditions of this study, the NOAEL for chronic toxicity was considered to be 350 ppm for male and female mice which corresponds to 47.2 mg/kg bw/day for males and 65.1 mg/kg bw/day for females. This value is based on treatment-related reductions in body weights, body weight gain and food consumption.

Overall, the test item was not carcinogenic in both species investigated (rats and mice).