thiacloprid (ISO);(Z)-3-(6-chloro-3-pyridylmethyl)-1,3-thiazolidin-2-ylidenecyanamide;{(2Z)-3-[(6-chloropyridin-3-yl)methyl]-1,3-thiazolidin-2-ylidene}cyanamide

Carcinogenicity

Description of key information

Rats

Key, M-003817-02-1, OECD 453, rat, 2 years,
NOAEL (general toxicity): 25 ppm (males, corresponding to 1.2 mg/kg bw/day) and 50 ppm (females, corresponding to 3.3 mg/kg bw/day)
LOAEL (general toxicity): 50 ppm (males, corresponding to 2.5 mg/kg bw/day) and 500 ppm (females, corresponding to 33.5 mg/kg bw/day)
NOAEL (carcinogenicity): 50 ppm (corresponding to 2.5 mg/kg bw/day in males and 3.3 mg/kg bw/day in females)
LOAEL (carcinogenicity): 500 ppm (corresponding to 25.2 mg/kg bw/day in males and 33.5 mg/kg bw/day in females)

 

Mice

Key, M-003819-02-1, OECD 451, mouse, 2 years,
NOAEL (general toxicity): 30 ppm (corresponding to 5.7 mg/kg bw/day in males and 10.9 mg/kg bw/day in females)
LOAEL (general toxicity): 1250 ppm (corresponding to 234.1 mg/kg bw/day for males and 475.3 mg/kg bw/day for females)
NOAEL (carcinogenicty): 30 ppm (females, corresponding to 10.9 mg/kg bw/day) and 2500 ppm (males, corresponding to 546.4 mg/kg bw/day)
LOAEL (carcinogenicty): 1250 ppm (females, corresponding to 475.3 mg/kg bw/day)

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:

14 Mar 1995 - 7 Apr 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

adopted 2018

Deviations:

yes

Remarks:

Mainly missing examinations, namely blood and urine were not withdrawn after 3 months and animals were not fasted prior to blood sampling, thyroid and uterus weight were not assessed, no skin histopathology, the feed was not analyzed for phytoestrogens

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

adopted 1981

Deviations:

no

GLP compliance:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan-Winkelmann, Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 5 - 6 weeks
- Weight at study initiation: 94 -141 g (males), 79 - 113 g (females)
- Fasting period before study: not applicable
- Housing: individually under conventional conditions in type IIa polycarbonate cages on low-dust wood granulate (Ssniff Spezialdiäten GmbH, Soest, Germany)
- Diet: fixed-formula standard diet (Altromin® 1321 powder supplied by Altromin GmbH, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 8 days

DETAILS OF FOOD AND WATER QUALITY: feed and water were regularly checked for contaminations.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 5
- Air changes (per hr): approx. 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 16 Mar 1995 To: 7 Apr 1997

Route of administration:

oral: feed

Vehicle:

peanut oil

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): fixed-formula standard diet (Altromin® 1321 powder supplied by Altromin GmbH, Lage, Germany). The test substance was blended (using a mixing granulator) with Altromin 1321 meal. To all diet mixtures including the control, peanut oil (DAB 19, 10 g per kg feed) was added to minimize dust formation.
- Storage temperature of food: room temperature

VEHICLE
- Justification for use and choice of vehicle: peanut oil (DAB 10) was mixed in the feed to prevent dust-formation.
- Amount of vehicle in feed: 1%

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Data on homogeneity and stability of the test substance in the administration vehicle covering the concentration range used were obtained before start of this study and homogeneity and stability were verified within a concentration range of 10 ppm to 20000 ppm. Under the sample preparation and handling conditions employed, stability in the diet was assured for a period of at least 14 days.

The test substance content of the diet mixtures was checked several times during the study (at least at start and end of the study, and several times during the study). For that purpose, samples of food mixes per dose were taken at the start of the feeding period and again at the end of the period after being kept under animal room conditions. All samples taken were kept deep frozen (<= -15°C) until examination.
Analyses were conducted using a HPLC method.
The test material content in the diet mixtures fed to the animals, prepared during the study, agreed with the target concentrations within defined limits. Recovery was between 48 and 137% for the 25 ppm samples, between 60 and 120% for the 50 ppm samples, between 88 and 114% for the 500 ppm samples, and between 83 and 113% for the 1000 ppm samples. As the recoveries determined for the two lower concentrations (25 and 50 ppm) were in some cases not valid, the procedure was adapted. Measurements using the new procedure were in agreement with the target concentrations.

Duration of treatment / exposure:

107 weeks (54 - 55 weeks for interim sacrifice)

Frequency of treatment:

continuously via the diet

Post exposure period:

no

Dose / conc.:

25 ppm (nominal)

Remarks:

corresponding to 1.2 and 1.6 mg/kg bw/day actual dose ingested (males and females, respectively)

Dose / conc.:

50 ppm (nominal)

Remarks:

corresponding to 2.5 and 3.3 mg/kg bw/day actual dose ingested (males and females, respectively)

Dose / conc.:

500 ppm (nominal)

Remarks:

corresponding to 25.2 and 33.5 mg/kg bw/day actual dose ingested (males and females, respectively)

Dose / conc.:

1 000 ppm (nominal)

Remarks:

corresponding to 51.7 and 69.1 mg/kg bw/day actual dose ingested (males and females, respectively)

No. of animals per sex per dose:

50/10 (interim sacrifice after 1 year)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose selection was based on results of a previously conducted subchronic toxicity study in Wistar rats (M-000863-01-1) in which rats received 0, 25, 100, 400 or 1600 ppm of the test substance in the diet. Body weight and body weight gain was reduced in 1600 ppm males and females. At 400 and 1600 ppm, liver enzymes were induced markedly, resulting in histopathological and macropathological changes (moderate hepatocellular hypertrophy). Therefore, 0, 25, 500 and 1000 ppm were administered in this study.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once daily on weekends and public holidays

DETAILED CLINICAL OBSERVATIONS: Yes
A detailed weekly report on the condition of the individual animals assessed the following: body surfaces and orifices, posture, general behavior, breathing and excretory products.

BODY WEIGHT: Yes
- Time schedule for examinations: Week 0-13 weekly; Week 14-105 every two weeks. Furthermore, body weights were recorded immediately before scheduled necropsy, for calculation of relative organ weights.

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
- Time schedule for examinations: Week 1-13 weekly; Week 14-105 every 4 weeks

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: No

WATER CONSUMPTION AND COMPOUND INTAKE : Yes
- Time schedule for examinations: Week 1-13 weekly; Week 14-105 every 4 weeks

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before start of treatment (Week 0), after 12 months and at the end of treatment (Week 103/105)
- Dose groups that were examined: pre-treatment and before necropsy after 2 years (all animals), after 1 year (control and highest treatment group)
- parameters checked: pupillary reflex of both eyes was first tested in a darkened room. After dilating the pupils with Mydriaticum-Stulln® drops, the refractive elements of the eye and the fundus were examined using an indirect ophthalmoscope. The eyes were also examined using a photo-slit lamp.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Weeks 26/29* (*when samples from Week 26 were found to be partly clotted, they were replaced with samples from Week 29), 53, 78 and 105
- Anesthetic used for blood collection: No (for blood withdrawal from the caudal veins for measurement of glucose concentration) Yes (diethyl ether, for blood withdrawal from the retro-orbital venous plexus for measurement of other parameters)
- Animals fasted: No
- How many animals: 10 animals per group
- Parameters checked: differential blood count, erythrocyte morphology (= red blood cell morphology), erythrocyte count (= red blood cell count; ERY), hemoglobin concentration in the blood (HB), hematocrit (= packed cell volume; HCT), leukocyte count (= white blood cell count; LEUCO), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular cell volume (MCV), thrombocyte count (= platelet count; THRO), thromboplastin time (Hepato-Quick; HQUICK)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Weeks 26, 53, 78 and 105
- Animals fasted: No
- How many animals: 10 animals per group
- Parameters checked alanine aminotransferase (ALAT), alkaline phosphatase (APh), aspartate aminotransferase (ASAT), gamma-glutamyltransferase (GGT), albumin (ALB), bilirubin (BILI-t), cholesterol (CHOL), creatinine (CREA), total protein (PROT), triglycerides (TRIGL), urea (UREA), glucose (GLUCOSE), chloride (Cl), calcium (Ca), inorganic phosphate (P), potassium (K), sodium (Na), triiodothyronine (T3), thyroxine (T4), thyroxine-binding capacity (TBC), thyroid stimulating hormone (TSH)
- Time schedule for liver investigations: Weeks 54/55
- How many animals: 10 rats/sex at 0 and 25 ppm and 5 rats/sex at 50, 500 and 1000 ppm.
- parameters checked: Cytochrome P-450 monooxygenases: 7-ethoxycoumarin-deethylase (ECOD), 7-ethoxyresorufin-deethylase (EROD), aldrin-epoxidase (ALD); Phase Il-enzymes: epoxide hydrolase (EH), glutathione S-transferase (GST), UDP-Glucuronyl-transferase (UDP-GT)

URINALYSIS: Yes
- Time schedule for collection of urine: Weeks 25, 52, 77 and 104 (over 16 h)
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes, but water was supplied
- parameters checked: blood, bilirubin, glucose, ketone bodies, pH, protein, sediment, urobilinogen, protein (PROT), volume (VOL), calcium (Ca), inorganic phosphate (P), potassium (K), sodium (Na), density

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

All animals were killed by exsanguination under diethyl ether anesthesia, necropsied and their organs and tissues subjected to thorough gross pathological examination. Animals that died spontaneously or were sacrificed in a moribund state during the study, were dissected at the earliest opportunity.

GROSS PATHOLOGY: Yes

Organ weights were recorded for brain, heart, liver, spleen, kidneys (both), adrenal glands (both), ovaries and testes (both)


HISTOPATHOLOGY: Yes
- fixed organs: Adrenals, aorta, brain (cerebrum, cerebellum, pons/medulla), cecum, colon, duodenum, epididymides, esophagus, eyes (with eyelids), exorbital lacrimal glands, femur (incl. bone marrow and knee joint), harderian glands, head-nose-pharynx area, heart, ileum, jejunum, kidneys, larynx, liver, lungs (prefixation by instillation with 4% buffered formaldehyde solution), lymph nodes (mandibular and mesenteric), mammary glands, optic nerves, ovaries (incl. oviduct), pancreas, physical Identifier (tattooed ears), pituitary, prostate, rectum, remaining intestine, salivary glands, sciatic nerve, seminal vesicles (with coagulating glands), skeletal muscle, skin (mammary region), spinal cord (cervical, thoracal, lumbar), spleen, sternum (with bone marrow), stomach (forestomach and glandular stomach), testes, thymus (if present), thyroid (with parathyroids), tongue, trachea, ureter, urethra, urinary bladder (prefixation by instillation with 4% buffered formaldehyde solution), uterus (with cervix), vagina, Zymbal glands and all tissues showing abnormalities

Staining: hematoxylin and eosin (H&E)
- embedding media: paraplast
- tissues: all organs except eyelids, head, larynx, Zymbal glands, remaining intestinal tissue, ureters, urethra, and physical identifier
- section thickness: 5 µm (4 µm for the uterus of the intermediate group females)
- animals investigated: All main groups were examined histopathologically (all organs listed above from all animals of all dose groups).
From the animals scheduled for interim sacrifice all blocked organs were examined in control and high-dose group. In the remaining groups after interim sacrifice, only the following organs were examined: lungs, liver kidneys, heart, thyroids, parathyroids, esophagus, trachea, and all gross findings.

Staining: Oil Red O
- animals: interim sacrifice animals
- tissues: cryo-cuts obtained from the formalin-fixed livers

In an amendment to the report, the uterus (horns and cervix) of the 25 and 500 ppm interim groups were also evaluated histopathologically.

Other examinations:

no

Statistics:

The statistical evaluation of data related to clinical chemistry, hematology, survival, body and organ weights as well as feed and water intake was performed using SAS® routines.

For continuous data that is presumed to be normally distributed with equal variances, an ANOVA followed by the Dunnett test was used. If heteroscedasticity appeared more likely a p value adjusted Welch test was applied. If no parametric analysis could be done, Kruskal-Wallis test followed by adjusted Mann-Whitney-Wilcoxon (U tests) was performed.

For global tests, multiple comparison procedures were used (two-sided).

Significant differences from the control group are indicated with "+" for p < 0.05 and "++" for p < 0.01.

Clinical signs:

no effects observed

Description (incidence and severity):

No differences between control and treatment groups were observed.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

mortality observed, non-treatment-related

Description (incidence):

During the course of the study, no evidence of a test substance-related increase in mortality was found. The incidences of death in control females was rather high. Cumulative mortalities were 11-12-14-10-9 and 28-22-14-19-17 for males and females, respectively (0-25-50-500-1000 ppm).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 25 and 50 ppm: There were no effects on body weights observed up to and including 50 ppm in males and females.
- 500 ppm: during the first 10 weeks of the study body weights in males were statistically significantly lower than in the corresponding control (males, Week 1-10, up to -7%), statistically significantly lower body weights than controls were observed in females starting at Week 8 (continued throughout the study, up to -15%).
- 1000 ppm: statistically significantly reduced body weight in males and females to the control, in males the difference was lower and fluctuated between -5% and - 12% throughout the study, with -9% at termination, in females reduced body weight became statistically significant in Week 5 and stayed reduced throughout the study (up to -21%), all compared to controls.

Summarized data can be found in Attachment 1 in the attached background material.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- 25 and 50 ppm: There were no effects on food consumption observed up to and including 50 ppm in males and females.
- 500 ppm: food consumption was reduced at several intervals during the study (slightly but statistically significantly) compared to controls.
- 1000 ppm: food consumption was reduced at several intervals during the study (slightly but statistically significantly) compared to controls.

Summarized Data can be found in Attachment 2 in the attached background material.

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

no effects observed

Description (incidence and severity):

No differences between control and treatment groups were observed.

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

Interim examination: No differences were found after one year between control and treatment groups.

Terminal examination:
- 1000 ppm: higher incidence of lens alterations in the cortex/nucleus (females) compared to controls.

Not treatment related findings
At 50 ppm and above, males showed increased cortical water clefts. Cortical or nuclear lens opacity incidences of all degrees were found in males in all treatment groups. However, both of these signs are common in aging rats. There was no dose-dependency observed when assessing both lesions together (17-31-37-23-23%). Therefore, these lesions were not considered treatment related. The incidence of lens alterations in the cortex/nucleus in females at 25 ppm was already higher than controls (incidence 2-15-13-18-32%). However, the controls were very low which could be due to the fact that the survival rate in control females was lower than in treatment groups. Moreover, up to 500 ppm, no dose-dependency could be established and the values were still within biological variance. Therefore, only for the high-dose females a treatment-related effect was considered. Similarily, the incidences of opacities in the retrolenticular area were increased in treatment groups of 50 ppm and higher in females (incidences 2-0-13-11-14%) but control and 25 ppm values were very low, no clear dose-dependency was found and males exhibited equally distributed incidences at higher degrees (26-27-20-25-26%). Therefore, this finding was not considered treatment-related.

Summarized data can be found in Attachment 3 in the attached background material.

Haematological findings:

no effects observed

Description (incidence and severity):

Several values were significantly different to control values at different time points, but no dose- or time-dependency could be established and/or the difference was not of biological significance.

Summarized data can be found in Attachment 4 in the attached background material.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- 25 and 50 ppm: There were no effects on clinical biochemistry observed up to and including 50 ppm in males and females.
- 500 ppm: slightly decreased triglycerides (females, all dates), slightly decreased total bilirubin (males, all dates), all compared to controls.
- 1000 ppm: increased ASAT and APh (Week 53, males), slightly increased cholesterol (Week 26 in males, females), slightly decreased triglycerides (females, all dates), slightly decreased total bilirubin (males, all dates), increased total protein (males and females, all dates), all compared to controls.

In blood electrolyte concentrations, no toxicologically relevant differences occurred. If values differed significantly from controls, these findings were sporadic, not dose-dependent and/or within the biological deviation.

Thyroid parameters:
- 500 ppm: increased TSH in some males (Week 26) compared to controls.
- 1000 ppm: non-significant and mostly slight increase in the TSH concentration (males, all dates with a peak in Week 26), statistically significantly increased TSH (Week 26 and 105, females), all compared to controls.

Enzyme activities in the liver
- 50 ppm: increased ECOD (males), ALD (males), EH (females), all compared to controls.
- 500 ppm: increased ECOD (males/females), ALD (males/females), EH (females), GST (males/females), UDP-GT (males/females), all compared to controls.
- 1000 ppm: increased ECOD (males, up to a factor of 4/females, up to a factor of 3.4), ALD (males, up to a factor of 2/females, up to a factor of 2.5), EH (males, up to a factor of 2/females, up to a factor of 3.3), GST (males, up to a factor of 1.5/females, up to a factor of 1.6), UDP-GT (males, up to a factor of 1.9/females, up to a factor of 2.5), all compared to controls.

In case of the 25 ppm dose level, ECOD and ALD were induced in males and EH in females. To test the validity of these findings, additional liver samples were analyzed (Attachment 6, Table 6 (b)) that could not support these data. Therefore, the increased ECOD, ALD and EH activity in males and females at 25 ppm compared to controls is seen as incidental. At the higher dose levels, the determination of enzymes in liver homogenates revealed an induction of all measured enzymes (except EROD) in male and female rats.

Summarized data can be found in Attachment 5 (clinical chemistry), Attachment 6 (thyroid parameters) and Attachment 7 (liver enzyme activities) in the attached background material.

Endocrine findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No differences between control and treatment groups were observed for blood, bilirubin, glucose, protein, urobilinogen, ketone, pH and sediments. Several quantitative parameters (density, K, Ca) were statistically significantly different to control values at different time points, but no dose- or time-dependency could be established and/or the difference was not of biological significance.

Summarized data can be found in Attachment 8 in the attached background material.

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, treatment-related

Description (incidence and severity):

Interim sacrifice:
- 25, 50 and 500 ppm: There were no effects on organ weights observed up to and including 500 ppm in males and females.
- 1000 ppm: significantly increased relative liver weights (+16%, females) compared to controls. There was also a trend observed in males (+9%) without reaching statistical significance.

Terminal sacrifice:
- 25, 50 ppm and 500 ppm: There were no effects on organ weights observed up to and including 500 ppm in males and females.
- 1000 ppm: increased absolute (males) and relative (males/females) liver weights compared to controls. Relative liver weights were increased by 31% in males and by 16% in females.

All other differences were not considered of biological significance as they did not show dose-dependency.

Summarized data can be found in Attachment 9 (interim sacrifice) and 10 (terminal sacrifice) in the attached background material.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Interim sacrifice: No differences were found between control and treatment groups.

Terminal sacrifice:
- 25 and 50 ppm: There were no effects observed up to and including 50 ppm in males and females.
- 500 ppm: increased incidence of cysts in the liver (females), thyroids showed enlargements and nodules (males, females), decreased incidence of nodules in the pituitary gland (females), slightly higher incidence of turbidity in the eye (females), all compared to controls.
- 1000 ppm: increased incidence of area / s (males) and cysts (females) in the liver, thyroids showed enlargements and nodules (males, females), increased incidence of nodules in the uterus, decreased incidence of nodules in the skin and in the pituitary gland (females), slightly higher incidence of turbidity in the eye (females), all compared to controls.

In an amendment to the study report the additional pathological and histopathological examination of the uterus in females of all dose groups from the interim sacrifice (previously only control, high dose group and gross abnormalities) was provided. There was no evidence of any gross finding in the uterus related to treatment with the test compound.

Summarized data can be found in Attachment 11 (terminal sacrifice) in the attached background material.

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Interim sacrifice:
- 25 and 50 ppm: There were no effects on organ weights observed up to and including 50 ppm in males and females.
- 500 ppm: hepatocellular hypertrophy (5/10 males and 5/10 females), focal fat infiltration (5/10 males), hypertrophy in the thyroid (7/10 males), colloidal clumping in the thyroid (10/10 males)
- 1000 ppm: hepatocellular hypertrophy (9/10 males, 8/10 females), focal fat infiltration (7/10 males), hypertrophy in the thyroid (8/10 males, 4/10 females), colloidal clumping in the thyroid (10/10 males, 8/10 females)

Summarized data can be found in Attachment 12 in the attached background material.

Final necropsy:
- 50 ppm:
Eyes: significant increase in retinal atrophy compared to controls (females).
Liver: hepatocellular cytoplasmic change (eosinophilic cytoplasm with basophilic strands) and predominantly centrilobular hepatocellular hypertrophy (males), increased mixed eosinophilic-clear cell foci in the liver (males), all compared to controls.
Thyroid: increased number of animals with hypertrophy of the follicular epithelium of the thyroid (males) compared to controls.
- 500 ppm:
Neurology: slightly increased radiculoneuropathy (females), sciatic nerve degeneration (males, females), all compared to controls.
Skeletal muscle: increased atrophy in skeletal muscles (females) compared to controls.
Eyes: significant increase in retinal atrophy (females), increased degeneration of lens fibers (cataract) in females, all compared to controls.
Liver: hepatocellular cytoplasmic change (eosinophilic cytoplasm with basophilic strands) and predominantly centrilobular hepatocellular hypertrophy (males, females), increased mixed eosinophilic-clear cell foci in the liver (males), slightly increased hepatic focal necrosis and biliary cysts (females), decreased incidence of basophilic foci (males, females), reduced clear cell foci (females), all compared to controls.
Brain: increased incidence of cholesterol clefts in the space between the anterior pituitary and the pars intermedia (males).
Thyroid: increased number of animals with hypertrophy of the follicular epithelium of the thyroid (males, females), thyroid colloid alteration and pigment in the follicular epithelium (males, females), all compared to controls.
Female reproductive tract: higher incidences of ovarian cysts (females) compared to controls.
- 1000 ppm:
Neurology: increased cholesterol clefts in the nerve roots of the spinal cord (females), significantly increased radiculoneuropathy (females), sciatic nerve degeneration (males, females), all compared to controls.
Skeletal muscle: increased atrophy, degeneration and mononuclear infiltrates in skeletal muscles (females) compared to controls.
Eyes: significant increase in retinal atrophy (females), increased degeneration of lens fibers (cataract) in females, compared to controls.
Liver: hepatocellular cytoplasmic change (eosinophilic cytoplasm with basophilic strands) and predominantly centrilobular hepatocellular hypertrophy (males, females), increased incidences of hepatocellular vacuolation (males), slightly increased hepatic focal necrosis and biliary cysts (females), increased mixed eosinophilic-clear cell foci in the liver (males, females), decreased incidence of basophilic foci (males), reduced clear cell foci (females), all compared to controls.
Mesenteric: increased incidence of sinus histiocytosis in the mesenteric lymph node (females) compared to control.
Brain: increased incidence of cholesterol clefts in the space between the anterior pituitary and the pars intermedia (males), all compared to controls.
Thyroid: increased number of animals with hypertrophy of the follicular epithelium of the thyroid (males, females), thyroid colloid alteration and pigment in the follicular epithelium (males, females), all compared to controls.
Female reproductive tract: higher incidences of ovarian cysts (females), higher incidence of lacteal cysts and galactocele in mammary glands (females), all compared to controls.

The additional evaluation of the uterus in the Amendment I showed that glandular hyperplasia of the uterine mucosa was found more frequently and to higher degrees in treated females (500 and 1000 ppm) compared to controls. However, it is not clear whether this finding is related to treatment or to other findings in the uterus like endometritis or stromal polyps which are frequently found in females of this strain and age and were also found in this study. Namely, endometritis was seen in one control and one female of the 500 ppm group, a stromal polyp occurred in an animal treated with 50 ppm and one female receiving 500 ppm had endometritis and a stromal polyp. Therefore, this finding is ambiguous.

As stated in the expert statement addressing target-organ findings after repeated exposure (M-282359-01-1), sciatic nerve degeneration is a common finding in aging rats and the muscle degeneration, which appears also spontaneously in untreated rats of high age, are most probably secondary to the changes in the nerve roots and in the sciatic nerve. Historical control data for sciatic nerve degeneration can be found in Attachment 18. Moreover, none of the observations was found at interim necropsy and other studies using even higher doses could not reproduce neurotoxicity. Therefore, a primary neurotoxic effect is unlikely.

The eye finding retinal atrophy is frequently observed as a spontaneous lesions in rats from control groups. In addition, the incidences are varying throughout the control groups. This can be seen from the table of incidences of retinal atrophy in control animals of chronic rat studies conducted at the testing laboratory in a similar time period (50 males and 50 females per group), which can be found in Attachment 18. Comparison of the incidences in the present study with the historical control data reveals that for retinal atrophy with control incidences of 3 - 21 per 50 animals in male rats and 12 - 29 per 50 animals in female rats, only the incidence at the highest dose exceeds the historical range slightly.

These non-neoplastic changes were considered as treatment-related. The hepatocellular hypertrophy is a known adaptive response giving evidence of liver enzyme induction. Summarized data can be found in Attachment 13 and 14 (additional uterus investigations) in the attached background material.

Several other obvious incidence differences, especially between high dose groups of interim and final sacrifices and controls may be partly related to reduced body weights/food consumption. These were reduced incidence of periarteritis in different organs and chronic progressive nephropathy in females, reduced incidence of medullary hyperplasia in the adrenals of males or reduced incidence of inflammatory infiltrates in the extraorbital lacrimal glands of females). They were considered secondary effects and not relevant for toxicological evaluation.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Interim necropsy: There were no stand-alone findings for neoplasia, but at 1000 ppm in males, one follicular cell adenoma in the thyroid was found, which is in line with the findings at terminal sacrifice.

Summarized data can be found in Attachment 15 in the attached background material.

Terminal sacrifice
- 25 and 50 ppm: There were no differences observed regarding neoplastic findings up to and including 50 ppm in males and females.
- 500 ppm: increased incidence of follicular cell adenoma of the thyroids (males), increased incidence of uterine adenocarcinoma (females) compared to controls.
- 1000 ppm: increased incidence of follicular cell adenoma of the thyroids (males, slightly elevated also in females), increased incidence of uterine adenocarcinoma compared to controls.

Other neoplasmas were decreased in treatment groups. Benign medullary tumors (phaeochromocytomas) of the adrenal glands occurred at lower incidence in males at 500 and 1000 ppm compared to controls. In females, incidence of fibroadenomas of the mammary gland and C-cell adenomas of the thyroid gland was reduced (1000 ppm) compared to controls.

Summarized results can be found in Attachment 16 in the attached background material.

Number of animals with tumors
- 1000 ppm: There was a slight increase in the number of animals with neoplasms, those with more than one neoplasm, and the number of primary (benign) neoplasms in 1000 ppm males, which can easily be explained by the increased thyroid follicular cell adenoma incidence. In females there was an increase in the number of animals with metastasising malignant tumors, which was caused by the higher uterine adenocarcinoma incidence.

Summarized results can be found in Attachment 17 in the attached background material.

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

25 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects observed at this dose level.

Remarks on result:

other: corresponding to 1.2 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

50 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects occurred at this dose level.

Remarks on result:

other: corresponding to 3.3 mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

50 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical biochemistry

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

urinalysis

Remarks on result:

other: corresponding to 2.5 mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

500 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical biochemistry

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

Remarks on result:

other: corresponding to 33.5 mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Remarks:

carcinogenicity

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

Remarks on result:

other: corresponding to 25.2 mg/kg bw/day in males and 33.5 mg/kg bw/day in females

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

50 ppm

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 2.5 and 3.3 mg/kg bw/day for males and females, respectively.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 ppm

Organ:

thyroid gland

uterus

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Historical control data from 1995 and 1998 can be found in the attached background material (Attachment 19 and 20).

Conclusions:

In this study, the long term toxicity and carcinogenic effects of the test substance were investigated in rats. The study was conducted according to OECD guideline 453 with deviations and under GLP conditions. The deviations were missing examinations, namely blood and urine were not withdrawn after 3 months and animals were not fasted prior to blood sampling, thyroid and uterus weight were not assessed, no skin histopathology was performed and the feed was not analyzed for phytoestrogens. Nevertheless, the study meets the criteria of a key study and is valid.

After long-term administration of the test substance via the diet, hepatic enzyme induction, namely of 7-ethoxycoumarin-deethylase (ECOD), aldrin-epoxidase (ALD), epoxide hydrolase (EH), glutathione S-transferase (GST) and UDP-Glucuronyl-transferase (UDP-GT), was shown to be a toxicologically relevant effect in rats. Most enzyme activities were statistically significantly increased in males and females at 500 ppm and higher doses. In addition, the hepatic markers aspartate-aminotransferase (ASAT) and alkaline phosphatase (APh) were increased in males (Week 53). Macroscopically and histopathologically, the liver was altered, emphasizing the hepatic effect of the test substance. Secondary effects were thyroidal alterations, both non-neoplastic and neoplastic, probably due to increased T4 turnover followed by increased TSH release and thyroidal hyperstimulation. Tumors were also found in the uterus along with histopathological and macropathological observations. These findings may be considered to be secondary to the liver enzyme induction. Changes in liver enzymes as well as liver pathology in general were more pronounced in males, as were thyroidal tumors. In females the test item induced more pronounced effects on the nervous system and skeletal muscles. However, as discussed above, these were only relevant at the higher dose levels and can be seen as a secondary effect to changes in the nerve roots. Oculotoxic effects were mainly observed in females at the two highest dose levels (500 and 1000 ppm) and after life-long treatment only. The test substance may not directly induce cancer, but formation of cancer was observed at high doses of the test substance (500 ppm and 1000 ppm) probably as secondary effect to hepatic enzyme induction. The NOAEL for general systemic toxicity in males was set at 25 ppm and for females at 50 ppm (corresponding to 1.2 mg/kg bw/day and 3.3 mg/kg bw/day, respectively). Tumors were seen in males and females at 500 ppm (thyroid and uterine, respectively, corresponding to 25.2 and 33.5 mg/kg bw/day).

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 May 1995 - 26 Feb 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

adopted 2018

Deviations:

yes

Remarks:

The dose span between the low and middle dose is greater than 10 and was not justified, clinical chemistry was not assessed.

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

adopted 1981

Deviations:

no

GLP compliance:

yes

Species:

mouse

Strain:

B6C3F1

Details on species / strain selection:

SPF-bred mice

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: A/S Bomholtgard, Denmark
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 5-6 weeks
- Mean weight at study initiation: 21 g (males), 17 g (females)
- Fasting period before study: not applicable
- Housing: individually in Makrolon® cages type II on low-dust wood granules
- Diet: fixed-formula standard diet during acclimatization and Altromin 1321 meal during the study period (supplied by Altromin® GmbH, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: yes, but length not specified

DETAILS OF FOOD AND WATER QUALITY: food and water were peridodically checked for contaminations

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 5
- Air changes (per hr): approx. 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 09 May 1995 To: 28 May 1997

Route of administration:

oral: feed

Vehicle:

peanut oil

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: food rations were changed weekly
- Mixing appropriate amounts with: Altromin® 1321 meal (using a mixing granulator)
- Storage temperature of food: room temperature

VEHICLE
- Choice of vehicle: Peanut oil (DAB 10)
- peanut oil was added to reduce dust formation
- Concentration in vehicle: 1%

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Data on homogeneity and stability of the test substance in the administration vehicle covering the concentration range of 10 ppm to 20000 ppm were obtained. The analytical investigations demonstrated that the test substance was stable in the diet for at least 2 weeks. The test substance content in the diet mixtures was analysed by HPLC several times during the study period. All samples taken were kept deep frozen (at temperatures of <= -15°C) until examination. The analytical data verified that the test compound content agreed with the target concentrations (84 - 117% of target).

Duration of treatment / exposure:

107 weeks (interim sacrifice after 53/54 weeks)

Frequency of treatment:

continously via the diet

Post exposure period:

None

Dose / conc.:

30 ppm (nominal)

Remarks:

corresponding to 5.7 and 10.9 mg/kg bw/day actual dose ingested for males and females, respectively

Dose / conc.:

1 250 ppm (nominal)

Remarks:

corresponding to 234.1 and 475.3 mg/kg bw/day actual dose ingested for males and females, respectively

Dose / conc.:

2 500 mg/kg bw/day (nominal)

Remarks:

corresponding to 546.4 and 872.5 mg/kg bw/day actual dose ingested for males and females, respectively

No. of animals per sex per dose:

50 (for all doses), additional 10 animals/sex for vehicle control and high dose (2500 ppm) that were sacrificed after 1 year

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The doses were based on a subchronic toxicity study in which 10 animals per sex and dose received 50, 250, 1250 and 6250 ppm of the test substance for 14 weeks. No effects were observed at 50 ppm. At 250 ppm, liver function was affected in males and at 1250 and 6250 ppm in males and females. Affected parameters were for example reduced bilirubin, increased N-demethylase activity and P450 content or reduced cholesterol. Liver weights were increased (both sexes, 1250 and 6250 ppm) and adaptive hepatocellular hypertrophy was observed from 250 ppm onward in males and from 1250 ppm onward in females. Moreover, body weight was reduced in both sexes in the high dose group and food consumption increased in males at 1250 and 6250 ppm. Based on these results, the dietary concentrations for the carcinogenicity study were set at 30, 1250 and 2500 ppm.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once daily on weekends and public holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
- Parameters: body surfaces and orifices, posture, general behavior, breathing and excretory products

BODY WEIGHT: Yes
- Time schedule for examinations: until week 13 weekly, from week 15 until end of study every 2 weeks

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
- Schedule: until Week 13 weekly, from Week 17 until end of study every four weeks
- Feeding period: approx. 7 days

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: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weeks 53, 79 and 104/106
- Anesthetic used for blood collection: Yes, diethyl ether
- Animals fasted: No
- How many animals: 20 of each group (leucocyte count)
- Parameters checked: Leukocyte count (= white blood cell count; LEUCO), differential blood count

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

Necropsy was performed by exsanguination under diethyl ether anesthesia. Organs and tissues were subjected to thorough gross pathological examination. Interim sacrifices were performed after one year (only control and high dose treatments). Animals that died spontaneously or were killed in a moribund state were dissected at the earliest opportunity.

GROSS PATHOLOGY: Yes
Organ weights were recorded for brain, liver, spleen, heart, kidneys (both), adrenal glands (both), ovaries (both) and testes (both)

HISTOPATHOLOGY: Yes
- tissues were fixed in 10% buffered formaldehyde with the exception of one liver lobe and the lungs, which were fixed in a 4% buffered formaldehyde solution
- tissues collected: adrenals, aorta, brain (cerebrum, cerebellum, pons/medulla), cecum, colon, duodenum, epididymides, esophagus, eyes (with eyelids), exorbital lacrimal glands, femur (incl. bone marrow and knee joint), gallbladder, harderian glands, head-nose-pharynx area, heart, ileum, jejunum, kidneys, larynx, liver, lungs, lymph nodes (mandibular and mesenteric), mammary glands, optic nerves, ovaries (incl. oviduct), pancreas, physical identifier (tattooed ears), pituitary, prostate, rectum, remaining intestine, salivary glands, sciatic nerve, seminal vesicles (with coagulating glands), skeletal muscle, skin (mammary region), spinal cord (cervical, thoracal, lumbar), spleen, sternum (with bone marrow), stomach (forestomach and glandular stomach), testes, thymus (if present), thyroid (with parathyroids), tongue, trachea, ureter, urethra, urinary bladder, uterus (with cervix), vagina, zymbal glands, gross abnormalities

lungs and urinary bladder were instilled with 4% buffered formaldehyde solution

- Thickness of sections: approximately 5 µm
- staining: hematoxylin and eosin (H&E), liver cryocuts of interim necropsy were stained with oil red O (ORO)

Other examinations:

none

Statistics:

The statistical evaluation of data related to hematology, body and organ weights as well as feed intake was performed using SAS® routines.
Statistical evaluations on body weight and organ weight data were done using the Dunnett-test in connection with a variance analysis. A Kruskal-Wallis-Test with a Steel-Test was performed when data of feed and water intake were analyzed.

For descriptive parameters, mean and median were calculated.

For continuous data with normal distributions and equal variances, a Dunnett's test was performed. If heteroscedasticity appeared more, likely a p value adjusted Welch test was applied. If parametric analysis could not be performed and for discrete random variables, the Kruskal-Wallis test followed by adjusted Mann-Whitney-Wilcoxon tests (U tests) was done. For global tests, multiple comparison procedures were used.
Relative organ weights were submitted to a logarithmic transformation prior to the statistical analysis.
Significant differences from the control group were indicated with "+" for p< 0.05 and "++" for p < 0.01.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Only occasional and sporadic clinical signs were noted.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Occasional deaths occurred throughout the study and after blood sampling in all groups. For males, the death incidences (without the incidences due to blood sampling) were 3-11-3-4 (control-30-1250-2500 ppm) in the 107-weeks treatment groups and for females 15-19-17-17 (control-30-1250-2500 ppm).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 2500 ppm: reduction of up to -9% in males compared to controls, reaching statistical significance from about Week 31

Body weights were comparable with control animals for all treated females and in males up to and including 1250 ppm. Mean body weights in females (all dose groups) marked as statistically significantly different from control were considered not toxicologically relevant as sometimes body weights were higher than control (e.g. 2500 ppm, week 47 and 77) or have to be seen in the context of slightly lower body weights at test start (1250 and 2500 ppm).

Summarized results can be found in Attachment 1 in the attached background material.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

-2500 ppm: increase in absolute (10%) and relative (21%) intake in males compared to controls, indicating a reduced food efficiency

Average food intake was comparable to controls in males up to and including 1250 ppm and in all treated females.

Summarized data can be found in Attachment 2 in the attached background material.

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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, treatment-related

Description (incidence and severity):

- 1250 ppm: slightly but statistically significantly increased leukocyte counts (all parameters measured) in males compared to controls
- 2500 ppm: slightly but statistically significantly increased leukocyte counts (all parameters measured) in males compared to controls

Results can be found in Attachment 3 in the attached background material.

Not treatment related (all compared to controls)
- 30 ppm: decreased mean leucocyte counts (males, week 104/106, due to relatively high control values)
- 1250 ppm: increased leucocyte counts (Week 53, females, no dose and time dependency)
- 2500 ppm: increased leucocyte counts (Week 53 and 79, females, no dose and time dependency)

There were no toxicologically relevant differences in differential blood count in all animals as compared to controls, since the mean monocyte counts identified as statistically significantly different (females, Week 79; males, Week 104/106) did not show any dose dependency and were thus considered as toxicologically irrelevant.

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, treatment-related

Description (incidence and severity):

- 1250 ppm: increased liver weights (absolute 9%, relative 8% in males, absolute 11 %, relative 9% in females) at final necropsy compared to controls.
- 2500 ppm: increased absolute (80%) and relative (70%) adrenal weights at interim necropsy in females, no differences in adrenal weights compared to controls observed in females at final necropsy; increased liver weights (absolute 25% in males, 33% in females; relative 32% in males, 32% in females) at interim necropsy and at final necropsy (absolute 12%, relative 17% in males; absolute 29% in females; relative 23% in females), all compared to controls. The weights of brain, heart, spleen, kidneys, testes and ovaries were not different from those of age-matched control animals.

No differences regarding organ weights were observed between the control and 30 ppm group.

Summarized results can be found in Attachment 4 in the attached background material.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross pathology of mice sacrificed at interim necropsy revealed no treatment-related findings in both sexes.

At terminal sacrifice, an increased incidence of nodules at/in the ovaries was found (incidence of 1-4-4-6 for 0-30-1250-2500 ppm, females).

All other findings were incidental since they occurred sporadically or without any dose-relationship. This included for example changes in size, color or swellings of various organs.

Summarized data can be found in Attachment 5 in the attached background material.

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Interim sacrifice:
- 2500 ppm: centrilobular hepatocellular hypertrophy with an increase of hepatocellular fat storage (fatty change, intracytoplasmic vacuoles) and a decrease of sex-specific renal vacuoles (all males), hypertrophy of the X-zone in the adrenal cortex (all females), all compared to controls.

Final sacrifice:
- 1250 ppm: increase in the incidence of focal or diffuse vacuolisation of the adrenocortical X-zone (females), increased incidence of centrilobular hepatocellular hypertrophy (males/females), increased fatty centrilobular change (males), increased lipid vacuolisation in the medullary regions of mandibular and mesenteric lymph nodes (males/females), increased number of large, eosinophilic, luteinized cells in the ovarian stroma or the surrounding adipose tissue (females), all compared to controls.
- 2500 ppm: increased incidence of focal or diffuse vacuolisation of the adrenocortical X-zone (females), increased incidence of centrilobular hepatocellular hypertrophy (males/females), increased fatty centrilobular change (males/females), increased hepatocellular degeneration (males), increased necrosis in the liver (males/females), increased lipid vacuolisation in the medullary regions of mandibular and mesenteric lymph nodes (males/females), increased number of large, eosinophilic, luteinized cells in the ovarian stroma or the surrounding adipose tissue (females), all compared to controls.

No differences regarding histopathology were observed between the control and 30 ppm group.


Summarized data can be found in Attachment 6 in the attached background material.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At interim necropsy, an adenoma in the Harderian gland (female, 0 ppm) and a sarcoma of the skin (female, 2500 ppm) were observed. Both are known findings in control mice of this age and were thus considered to be of spontaneous origin.

Final sacrifice:
- 1250 ppm: increased incidence of ovarian luteomas (females, 5 vs 0 in control and 1 in 30 ppm group)
- 2500 ppm: increased incidence of ovarian luteomas (females, 6 vs 0 in control and 1 in 30 ppm group)

From the 6 ovarian luteomas observed at 2500 ppm 5 luteomas were benign and 1 was a malignant tumor). As the historical incidence range of luteomas is 1-3 in 50 untreated female mice (1 malignant and 2 benign luteomas), the incidence in the 30 ppm-group is well within the historical range for this finding.)

No other dose- or time dependency in the incidence of neoplastic changes was observed. No differences regarding neoplastic changes were observed between the control and 30 ppm group. Summarized data can be found in Attachment 7 (neoplastic changes), 8 (number of tumors and number of animals with benign and/or malignant tumors) and 9 (time-dependency) in the attached background material.

Other effects:

not examined

Description (incidence and severity):

not applicable

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

30 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were seen at this dose level.

Remarks on result:

other: corresponding to 5.7 mg/kg bw/day in males and 10.9 mg/kg bw/day in females.

Key result

Dose descriptor:

LOAEL

Remarks:

general 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

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: corresponding to 234.1 mg/kg bw/day in males and 475.3 mg/kg bw/day in females

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

30 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects were observed at this dose level.

Remarks on result:

other: corresponding to 10.9 mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Remarks:

carcinogenicity

Effect level:

1 250 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: neoplastic

Remarks on result:

other: corresponding to 475.3 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects were observed up to the highest dose tested.

Remarks on result:

other: corresponding to 546.4 mg/kg bw/day

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 250 ppm

System:

female reproductive system

Organ:

ovary

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Historical control data from 1995 and 1998 can be found in the attached background material (Attachment 10 and 11). 

Conclusions:

The study was performed according to OECD guideline 451 and under GLP. It was performed to assess the carcinogenic effects of the test substance in mice. Under the conditions of the test, 30 ppm (corresponding to 5.7 and 10.9 mg/kg bw/day in males and females, respectively) was the NOAEL for general toxicity for male and female mice. Liver effects were seen in male and female mice and included increased organ weight, hepatocellular hypertrophy, fat storage, necrosis and degeneration. In females, increased adrenal weight was associated with hypertrophy and vacuolisation of the cortical X-zone in females. The liver effects and the concomitant hormonal changes may have caused these adrenal changes by affecting the development of the hormone-dependent X-zone. The incidence of eosinophilic luteinised cells in the ovarian stroma or the surrounding adipose tissue and of ovarian luteomas was increased. The report considered these effects to be secondary to the known liver enzyme induction and the subsequent hormone imbalance.
Increased incidences of ovarian luteoma, in most cases a benign tumor, were noted after two years of treatment with 1250 and 2500 ppm in the diet. Therefore, the NOAEL for carcinogenicity was 10.9 mg/kg bw/day in female mice. The NOAEL for carcinogenicity in male mice was 2500 ppm (corresponding to 546.4 mg/kg bw/day).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

2.5 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The quality of the database is good comprising two carcinogenicity studies in rodent species (rat and mouse), according to OECD TG 453 and 451, respectively, under GLP conditions. Both studies are considered of reliable quality and validity, fulfilling the criteria of a key study. Thus, both are suitable for assessment of the present endpoint.

Organ:

ovary

thyroid gland

uterus

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 respective endpoint summary

Justification for classification or non-classification

After two years of treatment, uterine adenocarcinomas and follicular cell adenomas of the thyroids were found in rats, while mice showed incidences of ovarian luteoma and hypertrophy of the adrenal X-zone. The effects in mice on the adrenal glands and ovaries are considered to be secondary to the known liver enzyme induction and the subsequent hormone imbalance and are therfore considered irrelevant to humans. The same is considered for the follicular cell adenomas of the thyroids in rats. Uterine adenocarcinomas in rats could be a further secondary effect to general hormone imbalance caused by hepatic enzyme induction. Thus, hormonal imbalance could be demonstrated as a plausible mode of action, and in addition, a genotoxic mode of action could be excluded with sufficient certainty (For more details please refer to IUCLID section 7.6).
The available data on carcinogenicity meet the criteria for classification according to Regulation (EC) 1272/2008 and are therefore sufficient for classification of the test substance for carcinogenicity. The substance is listed in Annex VI of Regulation (EC) 1272/2008 (Index No. 613-325-00-3) as a suspected human carcinogen category 2 (Carc. 2; H351).

Additional information

Two key studies were conducted to assess the carcinogenic potential of the test substance, one in rats and one in mice. Both studies were performed under GLP conditions according to OECD guidelines.  

 

Carcinogenicity study in rats

In a two year study with male and female Wistar rats, chronic and carcinogenic effects of the test substance were assessed (M-003817-02-1). As in the other studies, rats received the test substance in the diet. The dose levels of 25, 50, 500 and 1000 ppm were based on a subchronic toxicity study conducted previously in the same strain (M-000863-01-1). Analytical verification of the test substance concentration in the diet was performed by HPLC and feed consumption determined the actual ingested doses to be 1.2, 2.5, 25.2 and 51.7 mg/kg bw/day for males and 1.6, 3.3, 33.5 and 69.1 mg/kg bw/day for females. Groups of 50 female and male rats were investigated over two years, while additional groups of 10 male and female rats were killed after one year (interim sacrifice). Animals were checked daily for signs of ill health and body weight was recorded for all groups weekly in the first 13 weeks, thereafter in every other week. Consumption of feed and water was documented and ophthalmological examinations were performed before the start of the treatment and prior to sacrifice for interim and final sacrifice groups. To investigate hematology, thyroidal parameters and clinical chemistry, blood was withdrawn from all animals after 26, 53, 78 and 105 weeks of treatment. The blood from the 26 weeks time point was partly clotted so that for some animals the blood collection had to be repeated in Week 29. Urine was collected over 16 hours in Week 25, 52, 77 and 104 and analyzed. All animals were killed by exsanguination under diethyl ether anesthesia, necropsied and their organs and tissues were subjected to thorough gross pathological examination. Histopathology was performed on various organs to detect neoplastic and non-neoplastic changes.

The study was conducted according to OECD guideline 453 with some deviations. Blood and urine were not withdrawn after 3 months and animals were not fasted prior to blood sampling, thyroid and uterus weight were not assessed, no skin histopathology was performed  and the feed was not analyzed for phytoestrogens. Thyroid and uterus weight were not assessed at necropsy but could have given further insight into the effects of the test substance. However, the study was valid and was performed under GLP conditions.

During the course of the study, no evidence of a test substance-related increase in mortality was found. The incidence of death in control females was rather high. Overall mortalities were 11-12-14-10-9  and 28-22-14-19-17 for males and females, respectively (0-25-50-500-1000 ppm). The low survival rate of control females led to several uncommonly low findings in the observations described below. Also, no clinical signs were recorded that were abnormal in animals treated with the test substance. It was found that body weight was significantly reduced in males and females from 500 ppm onward. This finding was more pronounced in females which showed body weight reduction of up to -21%. Feed consumption was only slightly (but statistically significantly) reduced at several intervals in males and females.

Regarding non-neoplastic changes in the eyes, the only finding of the ophthalmological examination at terminal sacrifice was a higher incidence of lens alterations in the cortex/nucleus of females of the 500 and 1000 ppm treatment groups. Histopathological examinations in these groups revealed retinal atrophy and degeneration of lens fibers (cataract) which are age-associated spontaneous changes which can be attributed to a high-dose effect.

In the 1000 ppm group, the incidence of sciatic nerve degeneration was found to be increased in males and females as compared to the control groups. However, comparison with historical control data as presented in an expert statement (M-282359-01-1) revealed that sciatic nerve degeneration is a common finding in rats of that age and the frequency was within the historical control range.

The hematological and histopathological investigations did not provide evidence of a test substance-related injury to the blood, the coagulation, and the blood forming tissues.

Liver enzyme induction was seen from 50 ppm in males and from 500 ppm onward. Several Phase I and II liver enzymes showed enhanced activity compared to control, i.a. UDP-Glucuronyl-transferase ( UDP-GT). In interim and in final sacrifice, hepatocellular hypertrophy was observed (from 500 ppm onward, at final sacrifice of males also in the 50 ppm treatment group). Histopathology of animals killed after two years of treatment also revealed cytoplasmic change in the hepatocytes (eosinophilic cytoplasm with basophilic strands) in both sexes. In males of the high dose group, increased incidences of hepatocellular vacuolation was observed and in females the incidence of hepatic focal necrosis and biliary cysts was slightly elevated at 500 ppm and above. Moreover, males treated with 50 ppm or higher and females treated with 1000 ppm of the test substance showed increased incidences of mixed eosinophilic-clear cell foci in the liver while basophilic foci (liver) were reduced in both sexes and clear cell foci (liver) were reduced in females of the high dose group. The histopathological findings are likely linked to induced hepatic enzyme activity and clinical findings such as slightly reduced ASAT (males, females) and APh (males) activities in blood plasma, essentially at 1000 ppm. In addition, cholesterol was increased in both sexes in Week 26 while triglycerides (females) and total bilirubin (males) were slightly decreased and total protein concentrations were increased (males, females). All these findings occurred in males and females at 500 and/or 1000 ppm, with the exception of bilirubin alteration, which was found in male animals only. Macropathological observations support the finding of hepatic toxicity as liver weight was increased for both sexes (relative weights up to 31% in males at terminal sacrifice, and ca. 16% in females at both necropsies).

As reported in the short term studies (for more information, please refer to IUCLID section 7.5), the thyroid was also affected by treatment with the test compound in this study. At interim sacrifice, one male of the high dose group had a follicular cell adenoma. This finding would not be of concern if isolated, however, at final necropsy, there was a significant and dose-dependent increase in follicular cell adenomas of the thyroids in 500 and 1000 ppm males and a very slight increase in 1000 ppm females. Histopathological investigations revealed hypertrophy of the thyroid epithelium associated with an increase of colloid clumping in males of the 500 and 1000 ppm treatment groups at interim sacrifice and at 50 ppm and above at final sacrifice. In females, this change was found at final sacrifice only and at 500 ppm and above. However, these alterations were only minimal to slight. Colloid alteration and pigment in the follicular epithelium were significantly increased in both sexes from 500 ppm on (final sacrifice). Such effects on the thyroid function with corresponding non-neoplastic and neoplastic sequelae are often observed in rats as secondary effects of enzyme induction in the liver. Especially the induction of UDP-Glucuronyl-transferase (UDP-GT) can lead to increased degradation of circulating thyroid hormones resulting in thyroidal hyperstimulation. Deviating from the results of earlier short-term studies, no alterations in T3 and T4 levels were observed in peripheral blood but TSH was increased in males (500 and 1000 ppm) and females (1000 ppm), which may be an indication that the higher metabolic degradation of thyroid hormones may have been compensated by an increase in thyroid activity. At termination, enzyme levels in liver tissue were not investigated, but liver histopathology confirmed that thyroid findings were only seen at dose levels with liver enzyme induction (M-496853-01-1). Such activation may lead to the adenomas described and the increased incidence of thyroid adenomas may be a secondary effect of hepatic enzyme induction and not caused by a primary tumorigenic effect of the test item.

Further indications of secondary effects of the hormone imbalance caused by liver enzyme induction after dietary administration of the test compound, are effects on the female reproductive system. The incidences of ovarian cysts and uterine adenocarcinomas were increased from 500 ppm onward. The incidence of lacteal cysts in the mammary glands was reduced at a dose of 1000 ppm. Moreover, additional evaluation of the uterus after 1 year of treatment (interim sacrifice), showed that glandular hyperplasia of the uterine mucosa was found more frequently in treated females (500 and 1000 ppm) (Amendment I of report, 2007, M-003817-02-1). However, it is not clear whether this finding is related to treatment or to other findings in the uterus like endometritis or stromal polyps which are frequently found in females of this strain and age. Therefore, this finding needs to be assessed as ambiguous.

The substance also induced damage to the nervous system and skeletal muscles at 500 ppm and above in both sexes, although less pronounced in males. In females, increased incidence of cholesterol clefts in the nerve roots of the spinal cord (1000 ppm) was reported as well as radiculoneuropathy (500 and 1000 ppm), skeletal muscle atrophy (500 and 1000 ppm) and degeneration as well as mononuclear infiltrates in the sciatic nerve (1000 ppm). In both sexes, degeneration of the sciatic nerve was observed. However, sciatic nerve degeneration is a common finding in aging rats and muscle degeneration appears also spontaneously in untreated rats of high age. Moreover, none of the observations was found at interim necropsy and other studies using even higher doses could not reproduce neurotoxicty. Therefore, a primary neurotoxic effect is considered unlikely and the findings may be secondary to the general metabolic and hormonal imbalance caused by the substance via hepatic enzyme induction, the observed marked morphological changes of the liver and body weight reduction. The observed effects were therefore considered a high-dose phenomenon.

Further effects observed were higher incidences of cholesterol clefts between the anterior pituitary and the pars intermedia in males dosed with 500 and 1000 ppm and significantly increased incidences of sinus histiocytosis in the mesenteric lymph node of females at 1000 ppm.

Taken together the most sensitive parameter in this study was hepatic enzyme induction, especially of UDP-GT, in males from 50 ppm and in females from 500 ppm onward. Further phase I and II enzymes were also induced as well as the hepatic markers ASAT and APh in males. Macroscopically and histopathologically, the liver was altered, emphasizing the hepatic effect of the test substance. Secondary effects were thyroidal alterations, both non-neoplastic and neoplastic. Tumors were also found in the uterus along with histopathological and macropathological changes. These findings may be due to hormonal imbalance as a consequence of the described hepatic enzyme induction. Changes in liver enzymes as well as liver pathology in general were more pronounced in males, as were thyroidal tumors. In females the test item induced more pronounced effects on the nervous system and skeletal muscles. However, as discussed above, these were only relevant at the highest dose levels and can be seen as a high-dose phenomenon. Oculotoxic effect effects were observed in females of the highest dose levels and after life-long treatment only. Even though the test substance may not directly induce cancer, formation of cancer was observed at high dietary doses of the test substance (500 ppm and 1000 ppm). The NOAEL for carcinogenity for males and females was set at 50 ppm (corresponding to 2.5 mg/kg bw/day and 3.3 mg/kg bw/day, respectively).

 

As mentioned above, an additional expert statement (M-496853-01-1) investigated the mode of action of the development of thyroid tumors in male rats and their human relevance based on the available repeated dose data base. The author concluded that due to the increased activity of UDP-GT as a consequence of liver enzyme induction by the test compound, circulating T4 was increasingly metabolized and excreted via the bile. This led to elevated TSH release into the blood stream from the pituitary and activation of the thyroid. T3 and T4 levels were altered in some but not all repeated dose studies. However, in all studies, thyroid hormone determination was performed on maximal 10 animals per sex, which often leads to uncertainty in the results. It should also be noted, that TSH, T3 and T4 levels can change due to environmental factors and can also be influenced by the circadian rhythms of rats (Jordan et al., 1980).

All findings in the thyroid and on thyroid parameters were at doses associated with hepatic enzyme induction. At these dose levels, hepatocellular hypertrophy was also observed, always more pronounced in males than in females. It is commonly known that male rats in many cases display a more pronounced liver enzyme induction than female rats after administration with xenobiotics.

From the different studies conducted it is evident that the induction of hepatic enzymes is crucial for the secondary effects observed in rats. It was found by different studies, that human cytochrome P450 induction is lower than for rats (Silkworth et al., 2005; Waxman, 1999; Xu et al., 2000). Moreover, rats are more susceptible to thyroid cancer than humans (U.S. EPA, 1998, Hill et al., 1998; McClain, 1989). One reason for this is the shorter half-life of T4 in the rat due to the lack of high affinity thyroxine-binding globulin which is available in humans but missing in rats, leading to facilitated metabolism and excretion of T4. In addition, T3 has a shorter half-life in rats, which contributes together with the higher turnover of T4 to increased TSH production and release in rats compared to humans. Therefore, the thyroid at normal state is already more activated in rats compared to humans and the increased incidence of thyroid adenomas seen in the carcinogenic study may not be relevant to humans.

The mode of action observed and analyzed for the long-term study with the test substance in Wistar rats has been addressed in the Guidance on the Application of the CLP Criteria (version 5.0, 2017) by ECHA. In chapter 3.9.2.5.3. on mechanisms not relevant to humans, it was stated: "Humans, unlike rodents, possess a T4 binding protein that greatly reduces susceptibility to plasma T4 depletion and thyroid stimulation. Thus, such a mechanism/effect cannot be directly extrapolated to humans, i.e. these thyroid effects observed in rodents caused by an increase in hepatic UDP-GT are therefore considered of insufficient concern for classification."

 

Carcinogenicity study in mice

The long term and carcinogenic effects of the test substance were investigated in male and female B6C3F1 mice in a carcinogenicity study for 107 weeks with an interim sacrifice after 1 year according to OECD guideline 451 (M-003819-02-1). Groups of 50 male and female mice were fed with the test substance via the diet at doses of 0, 30, 1250 or 2500 ppm. For the interim sacrifice after one year, additional groups of 10 male and female mice received the vehicle control (plain diet with 1% of peanut oil) and the high dose. The actual ingested doses were determined as 5.7, 234.1 and 546.1 mg/kg bw/day for males and 10.9, 475.3, 872.5 mg/kg bw/day for females for the 107 weeks interval. Animals were checked daily for mortality and morbidity, body weight was recorded weekly until Week 13 and after that every other week, blood was withdrawn in Weeks 53, 79 and before terminal sacrifice for analysis of leucocyte count and differential blood count. At interim and terminal sacrifice, gross pathology was performed and various tissues were collected for histopathological analysis.

No treatment-related effects on mortality were found. Throughout the study and after blood sampling, occasional deaths occurred in all groups. For males, the death incidences were 3-11-3-4 (control-30-1250-2500 ppm) in the 107-weeks treatment groups and for females 15-19-17-17 (control-30-1250-2500 ppm). Clinical signs were also not observed after by treatment with the test substance.

Body weight was reduced up to 9% in males of the high dose group throughout the study. However, feed consumption in this group was increased by 10% (absolute intake). In relation to body weight the increase was 21%, suggesting a reduced feed efficiency. Females were not affected in body weight or feed consumption at any dose level. Regarding hematology, only males were affected. After treatment with 1250 and 2500 ppm, slightly but statistically significantly increased leukocyte counts for all parameters measured were observed. In females, only occasional findings were reported, that did not show dose- or time-dependency and were therefore not considered toxicologically relevant.

In the middle dose, both males and females showed increased liver weights at final necropsy compared to controls (the increase ranging from 9-12% and 11-29% of absolute liver weights in males and females and 8-17% and 9-23% of relative liver weight in males and females, respectively). Additionally, in the high dose group this increase was also seen at interim sacrifice (absolute weights increased by 25% in males, by 33% in females; relative weights increased by 32% in males, by 32% in females, as compared to controls). This corresponded with histopathological findings in the liver. At the final sacrifice, males and females receiving the middle and high dose showed increased centrilobular hepatocellular hypertrophy and increased incidences of necrosis in the liver. Increased fatty centrilobular change was seen in males of the middle and high dose group and females of the high dose group. At interim sacrifice, centrilobular hepatocellular hypertrophy with an increase of hepatocellular fat storage (fatty change, intracytoplasmic vacuoles) was found in males of the high dose group. These changes are probably associated with increased liver enzyme induction as seen in several studies.

The relative and absolute weight of adrenal glands was increased for females of the high dose group at terminal sacrifice (80 and 70%, respectively). Histopathological examinations showed hypertrophy of the X-zone in the adrenal cortex in all females of the high dose at interim sacrifice as well as an increase of focal or diffuse vacuolisation of the adrenocortical X-zone at terminal sacrifice in the middle and high dose. The adrenal changes may be secondary effects to the described liver enzyme induction and concomitant hormonal changes affecting the hormone-dependent X-zone. The X- Zone is a structure that is present in female mice but not in other laboratory animals or humans. The significance of the dose-related increase of X-zone vacuolation seen in the present studies is unclear but probably related to hormonal imbalances since development and disappearance of this zone is under hormonal influence.

Several effects were observed in the female reproductive organs, especially in and near the ovaries: an increased number of large, eosinophilic, luteinized cells in the ovarian stroma or the surrounding adipose tissue was found in the high dose group at terminal sacrifice as well as an increased incidence of ovarian nodules. The incidence of ovarian nodules was 1 in the control group and 4, 4 and 6 in the 30, 1250 and 2500 ppm groups, respectively. Moreover, the incidence of ovarian luteomas was statistically significantly increased in the mid and high dose group (5 and 6, respectively). In the female animals of the control groups, no case of ovarian luteoma was found and in the low-dose group at 30 ppm, only one incidence occurred, which is in the range of the historical control data. The report considered these effects to be secondary to the known liver enzyme induction and the subsequent hormone imbalance.

 

Overall, the carcinogenicity 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. Under the conditions of the test, 30 ppm was the NOAEL for general systemic toxicity for male and female mice (corresponding to 5.7 mg/kg bw/day in males and 10.9 mg/kg bw/day in females). Increased incidences of ovarian luteoma, in most cases a benign tumor, were noted after two years of treatment with 1250 and 2500 ppm in the diet. Therefore, the NOAEL for carcinogenicity was 10.9 mg/kg bw/day in female mice. The NOAEL for carcinogenicity in male mice was 2500 ppm (corresponding to 546.4 mg/kg bw/day).