Cyclopropanecarboxylic acid, [(3S,4R,4aR,6S,6aS,12R,12aS,12bS)-3-[(cyclopropylcarbonyl)oxy]-1,3,4,4a,5,6,6a,12,12a,12b-decahydro-6,12-dihydroxy-4,6a,12b-trimethyl-11-oxo-9-(3-pyridinyl)-2H,11H-naphtho[2,1-b]pyrano[3,4-e]pyran-4-yl]methyl ester

Carcinogenicity

Description of key information

The potential oncogenicity of the test substance was evaluated in oncogenicity studies in rats and mice.

In a 2-year carcinogenicity study with rats, the test substance was administered to groups of Fischer rats for 24 months, at dietary concentrations of 0, 100, 300 or 1000 ppm. No treatment-related abnormalities were noted in mortality, general clinical observation, hematology, organ weight and body weight ratio or gross pathology in any treated groups. Body weights in females in the 1000 ppm group were significantly lower than those in the control group at several test weeks. Food consumptions in females in the same group also decreased significantly for several weeks. Adenocarcinoma of the uterus in females increased significantly in the 1000ppm group, whereas the incidences in the treatment groups were unrelated to the dose levels and no significant differences of the incidences of the tumor were noted in all females in any treatment groups. The study concluded that there was no treatment related carcinogenic dose up to the maximum treatment level of 1000 ppm in males and females (male: 42.7 mg/kg/day, female: 50.8 mg/kg/day). A NOAEL for the study was not reported, but 300 ppm (12.9 for males and 15.5 mg/kg bw/d for females) is consistent with the reported data.

In a second supplemental 2-year carcinogenicity study with rats, the test substance was administered to groups of Fischer rats at dietary concentrations of 0, 1000 or 3000 ppm for a period of 2 years. There were no treatment-related clinical signs or neurobehavioral effects. Toxicity was observed in both sexes at both dose resulting in effects on bodyweight, food consumption, organ weights and gross pathology. Histopathological changes were observed in the liver and kidney. Adenocarcinoma of the uterus in females increased significantly in both the 1000 and 3000 ppm group. The NOAEL for all effects observed in this study was established in a prior rat cancer study.

In an 18-month carcinogenicity study mice (52/sex/dose), ICR [Crlj:CD1(ICR)] mice were given 0,120,700, 4000 (males) or 4000/3000/2000 ppm (females) of the test substance in the diet. At the high dose, treatment to females caused death or moribundity; low body weight, food consumption, and food efficiency; high total leukocyte, lymphocyte, and large unstained cell counts; effects on the spleen and heart; and several histopathological changes including vacuolation in multiple tissues, decreases of hematopoiesis in the bone marrow, and lymphoid depletion. At the dose level of 4000 ppm in males, low body weight, and the effects on the liver (centrilobular hepatocellular hypertrophy) and submandibular gland (depletion in granular ducts) were observed. The no-observed-adverse-effect level (NOAEL) in ICR [Crlj:CD1(ICR)] mice was considered to be 700 ppm (males, 78.7 mg/kg/day; females, 75.8 mg/kg/day) under the conditions of this study. There was no increase in incidence of neoplastic lesions in any treated groups of either sex. There were no increases of rare types of tumors, earlier occurrence of spontaneous neoplasm, or other sign indicating carcinogenicity.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From: July 5, 2011 To: March 6, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: JMAFF No 12 Nosan No 8147;

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.4200 (Carcinogenicity)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Lot number: 080722
Expiry: July 31, 2013
Appearance: Pale yellow green powder

Species:

rat

Strain:

Fischer 344/DuCrj

Details on species / strain selection:

The rat is one of animal species used generally for recommended carcinogenicity studies of agricultural chemicals, and abundant biological reference data on this strain have been accumulated in the testing facility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Source: Atsugi Breeding Center, Charles River Japan, Inc. (Kanagawa, Japan)
Age at study initiation: 5 – 6 weeks at dosing
Weight at dosing (Day 0): 102-120 g for males and 84-101 g for females
Fasting period before study: Not specified in the report
Housing: One or two animals of the same sex were housed in a stainless steel wire-meshed cage (width 21 cm x depth 35 cm x height 20 cm) during the quarantine and acclimatization period before grouping, and two animals of the same sex in each group were housed in the same cage during the treatment period after grouping. Cages and racks were exchanged once every 3 weeks.
Diet: A powdered basal diet for rats, Oriental Yeast Co., Ltd., Tokyo, Japan ad libitum
Water: Tap water (well water) sterilized with sodium hypochlorite, ad libitum
Acclimation period: 12 Days

ENVIRONMENTAL CONDITIONS
Temperature: 20-25 °C
Humidity: 30-70 %
Air changes: ≥10 times/hour
Photoperiod: 12 hours/day (light on at 07:00 and off at 19:00)

IN-LIFE DATES: From: July 19, 2011 To: December 26, 2013

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
To prepare the treated diets, 40 g and 120 g of test substance for the 1000 ppm and 3000 ppm groups were mixed with 1960 kg and 1880 kg of basal diets, respectively. Then the test substance was smashed and mixed gradually with small amount of the basal diet, and subsequently a total 2 kg of the preliminary mixed diet was shaken and mixed in a vinyl bag for approximately 5 minutes. Two lots of the preliminary mixed diet (2 kg) were prepared for each dose group.

Each test substance mixed diet was prepared one week and 2 weeks before the initiation of treatment in males and in females, respectively, and then at a 3 or 4-week-interval.

The preliminary mixed diets in each group were mixed with 38 kg of basal diets, and finally two lots of 40 kg of mixed diets were prepared for each dose group, using the mixer Mighty 120 (Aicohsha Manufacturing Co., Ltd., Saitama, Japan).

The test substance mixed diet in each group prepared was subdivided to 8 lots, approximately 10 kg in each, and tightly packed in a vinyl bag with a label indicating dose groups, preparation date and treatment weeks, put in a container and stored in a refrigerated stock room for diets and beddings under light-protected and refrigerated condition (actual temperature: 2-8 °C) until feeding.

For feeding, the test substance mixed diets, kept in the airtight containers, were brought from a refrigerated stock room for diets and beddings and then were kept in the animal room under light-protected and room temperature condition.

The test substance mixed diets were prepared one week before the treatment and 27 times during the test period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Three out of 5 samples (approximately 10 g each) collected from the test substance mixed diet in each treated group at the 1st, 7th, 14th, 21st and 27th (Final) preparations were analyzed for concentration and homogeneity of test substance. One sample from the basal diet in the control group was analyzed in the same manner. A part (approximately 10 g) of the residual mixed diets (the 1st, 7th, 14th, 21st and 27th prepared diets) in each group was collected after feeding and analyzed for stability of the test substance. The allowable range of the concentration of test substance in the test substance mixed diets was defined to be within ±15 % of selected dose levels. On the day of each measurement of test substance concentrations, the medium concentration (ST3: 0.5 mg/L) of the calibration curve was measured (n = 1) following the measurement of the test substance concentration for the quality control of measurement system. The allowable range of the measured values was defined to be within ± 5 % of the theoretical value (0.5 mg/L).

Homogeneity of test substance in the test substance mixed diets at the 1st, 7th, 14th, 21st and 27th preparations was also analyzed by using 3 out of 5 samples in each dose level. The coefficient of variation in all samples from each dose level was within 2.0 % at the 1st preparation, within 0.8 % at the 7th preparation, within 0.5 % at the 14th preparation, within 1.4 % at the 21st preparation and within 1.1 % at the 27th preparation. The homogeneity of test substance in the test substance mixed diets in all dose groups was adequate.

Stability of the test substance in the test substance mixed diets was analyzed by using a part (approximately 10 g) of the residual mixed diets (the 1st, 7th, 14th, 21st and 27th prepared diets) in each dose level. Those actual levels ranged from 97 % to 95 % of the selected dose levels at the 1st preparation, from 89 % to 93 % at the 7th preparation, from 96 % to 97 % at the 14th preparation, from 96 % to 97 % at the 21st preparation and 97 to 98 % at the 27th preparation being within the allowable range (± 15 %). The test substance was stable in the diet during the treatment period and the storage method of the test substance mixed diets was acceptable.

Duration of treatment / exposure:

2 years (104 weeks)

Frequency of treatment:

Daily

Post exposure period:

None specified

Dose / conc.:

0 ppm

Remarks:

plain diet

Dose / conc.:

1 000 ppm (nominal)

Remarks:

corresponding to 41.6 mg/kg bw/d in males, 50.4 mg/kg bw/d in females

Dose / conc.:

3 000 ppm (nominal)

Remarks:

corresponding to 128.2 mg/kg bw/d in males, 146.9 mg/kg bw/d in females

No. of animals per sex per dose:

50 animals/sex/dose

Control animals:

yes, plain diet

Details on study design:

DOSE SELECTION RATIONALE
The 2-year feeding study entitled of the test substance has been performed (xxxx). In the study, the test substance was mixed in the diet at 0ppm (control), 100ppm, 300ppm and 1000ppm and fed to 50 males and 50 females Fischer strain (F344/DuCrlCrlj) of rats in each dose group. The Dose Adequacy Review Team (DART) of the Health Effects Division of the US Environmental Protection Agency reviewed the dose levels in an ongoing 2-year rat cancer study and concluded that an additional dose of 3000ppm was required to meet the maximum tolerated dose (MTD) requirements. The agency also requested that the 1000 ppm dose be repeated to bridge the results to the ongoing rat cancer study that was being conducted at dose levels of 100, 300 and 1000 ppm (Reference Memorandum dated November 18, 2010; PC Code 036605; Decision No. 440705, TXR No. 0055537, DP Barcode D382921).

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS
Mortality: at least twice a day on weekdays and once a day on Saturdays, Sundays and holidays during the treatment period.
General clinical observation: once a day during the treatment period.

Inside of cage: Excitement, Sedation, Abnormal posture (prone position, lateral position, etc.), Abnormal behavior (moving backward, stereotypy, self-biting, etc.).
Handling: Positional passively (including response changes to stimulation), Changes of muscle tone (spasticity, flaccidity), Tremor, Palpebral closure, Salivation, Lacrimation, Discharge (auditory, nasal, vaginal, etc.), Exophthalmos, Changes of body temperature (hyperthermia, hypothermia), Abnormal breathing noise, Fur-appearance, Dermal and mucosal color.
Outside of cage: Jumping, Circling, Convulsion, Abnormal gait (coordination, staggering, dragging, hind limb paralysis, etc.), Locomotor activity (hyperlocomotion, hypolocomotion), Respiration (tachypnea, bradypnea), Vocalization, piloerection, Abnormal posture (prone position, lateral position, etc.), Abnormal behavior (moving backward, stereotypy, self-biting, etc.).

In addition, the observation for nodules including palpation was performed once a week.

BODY WEIGHTS
Body weights of all animals were measured on the day of animal receipt and on the grouping day. After grouping, body weights of the animals were measured once a week from test week 1 to 13 and once per 4 weeks from test week 16 to the termination of treatment. Final body weights of the animals were measured before euthanasia on each necropsy day and on the day at death.

FOOD CONSUMPTION
Food consumption (3-day or 4-day total amount) in each cage was measured once at test week 1 and twice a week during test week 2 to 13 and twice per 4 weeks from test week 16 to the termination of treatment. The total amount of food consumption was converted to a daily amount of one animal in each group. The weekly mean food consumption (g/rat/day) of males and females in each group was calculated based on the mean food consumption of each cage. The total mean food consumption of males and females in each group during the treatment period was calculated by averaging the weekly mean food consumption.

The mean test substance intake (mg/kg/day) of males and females in each dose-group was calculated in each measuring week according to the following formula:
Mean test substance intake = mean food consumption x selected dose level/mean group body weight.

In addition, the total mean test substance intake during the treatment period in males and females in each dose group was calculated by averaging the weekly mean test substance intake.

HAEMATOLOGY
Hematological examinations were performed on all animals killed as designed at each necropsy day (after the termination of 104-week treatment). The animals were anesthetized by diethyl ether inhalation and euthanized by blood collection from the abdominal artery. Simultaneously, blood smear slides of each animal were prepared and stained with May-Grünwald Giemsa. EDTA-2K was used as anticoagulant. The number of white blood cells in all animals killed as designed was measured by using an automated hematology analyzer. Differential leukocyte count in all animals killed as designed in the control and high dose groups was measured microscopically. However, differential leukocyte count in other dose groups was not examined because hematopoietic tumors related to the treatment with the test substance were not observed in the high dose groups.

At the termination of test week 52 and 78 (test week 53 and 79, respectively), small amounts of blood samples in all surviving animals were collected by cutting the tip of tail under anesthesia with diethyl ether inhalation, and blood smear slides were prepared and stained with May-Grünwald Giemsa. However, differential leukocyte count was not measured because hematopoietic tumors were not noted in relation with the test substance treatment in the any examinations after the termination of treatment.

Blood smear slides stained with May-Grünwald Giemsa were prepared from the blood of any animals in moribund state by cutting the tip of tail under anesthesia with diethyl ether inhalation during the treatment period. Differential leukocyte count was measured microscopically using the slides in moribund animals which were suspected to have hematopoietic tumors at necropsy. Individual values of differential leukocyte counts in those animals were only described in the final report and were not calculated.

Sacrifice and pathology:

NECROPSY
Necropsy was conducted on all dead animals, all moribund animals and all animals killed as designed. The moribund animals and animals killed as designed were anesthetized by isoflurane inhalation. The moribund animals were euthanized by bleeding from the neck artery, and the animals killed as designed with blood sampling were euthanized by blood collection from the abdominal artery.

ORGAN WEIGHTS
- absolute organ weights. Heart, Spleen, Liver, Kidneys (bilateral), Testes (bilateral), Epididymides (bilateral), Ovaries (bilateral), Uterus, Brain, Adrenal glands (bilateral).
- measured before fixation in all males and females killed as designed in each group.
- Relative organ weights were calculated as ratios of organ weights to final body weights.

HISTOPATHOLOGY
The following organs and tissues were removed from all animals at necropsy and fixed in 10 % neutral-buffered formalin except for the eyes and testes, which were fixed in Bouin solution. The lungs were infused with applied volume of 10 % neutral-buffered formalin from the trachea and then fixed in the fixative.
Heart, Aorta, Spleen, Thymus, Bone with bone marrow (sternum, right femur and knee joint), Mandibular lymph node, Mesenteric lymph node, Laryngopharynx, Trachea, Lungs with bronchi, Salivary glands (mandibular gland and sublingual gland), Tongue, Esophagus, Stomach (forestomach and glandular stomach), Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Liver, Pancreas, Kidneys (bilateral), Urinary bladder, Testes (bilateral), Epididymides (bilateral), Seminal vesicle and coagulation glands (bilateral), Prostate gland, Ovaries (bilateral), Uterus (including cervix), Vagina, Brain (cerebrum, cerebellum, pons and medulla oblongata), Vertebrae with spinal cord (cervical, thoracic and lumbar regions), Ischiadic nerve (right), Eyes (bilateral), Harderian glands (bilateral), Pituitary, Thyroids with parathyroids (bilateral), Adrenal glands (bilateral), Skeletal muscle (right femur), Skin (dorsal region), Mammary gland (the 2nd and 3rd glands), Skull with nasal tissue, Oral mucosa and tympanum, All gross lesions.

Histopathological examination was conducted on the following organs from all animals in all groups: Heart, Aorta, Spleen, Thymus, Bone with bone marrow (sternum, right femur and knee joint), Mandibular lymph node, Mesenteric lymph node, Nasal cavity, Laryngopharynx, Trachea, Lungs with bronchi, Salivary glands (mandibular gland and sublingual gland), Esophagus, Stomach (forestomach and glandular stomach), Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Liver, Pancreas, Kidneys (bilateral), Urinary bladder, Testes (bilateral), Epididymides (bilateral), Seminal vesicle and coagulation glands (bilateral), Prostate gland, Ovaries (bilateral), Uterus (bilateral horns and cervical portions), Vagina, Brain (cerebrum, cerebellum, pons and medulla oblongata), Vertebrae with spinal cord (cervical, thoracic and lumbar regions), Ischiadic nerve (right), Eyes (retina and optic nerve, bilateral), Harderian glands (bilateral), Pituitary, Thyroids with parathyroids (bilateral), Adrenal glands (bilateral), Skeletal muscle (right femur), Skin (dorsal region), Female mammary gland (the 2nd and 3rd glands), All gross lesions. The examination was carried out on paraffin sections stained with hematoxylin and eosin prepared by a routine method.

Statistics:

Body weight, food consumption, number of white blood cells, organ weights and organ weight to body weight ratio were assessed by Bartlett’s test (significance level: 5 %). Homogeneous data were assessed by one-way analysis of variance (p=0.05). If the result was significant, the data were assessed between the control group and the treated groups by Dunnett's multiple comparison test (two-tailed, p=0.05 and 0.01). If the variance was heterogeneous, the data were assessed by Kruskal-Wallis rank test (p=0.05). If the result was significant, the data were assessed between the control group and the treated groups by Dunnett type joint-ranking test (two-tailed, p=0.05 and 0.01 %).

Mortality, general clinical observations, gross pathological findings and histopathological findings were assessed between the control group and the treated groups by Fisher's exact probability test (one-tailed, p=0.05 and 0.01 %). However, the statistical evaluation of the histopathological findings was carried out between the control group and the high dose (1000ppm) group in the total number of all animals and animals killed by design. Those of the adrenal gland in male and the uterus in female were carried out between the control group and the treated groups.

Differential leukocyte count was assessed by F test (two-tailed, significance level: 5 %) between the control group and the high dose group. As the results, if the variance was homogeneous, the data were assessed by Student’s t-test (two-tailed, p=0.05 and 0.01 %). If the result was significant, the data were assessed by Wilcoxon test (two-tailed, p=0.05 and 0.01 %).

Mortality was assessed by the life table test. In the life table test, survival curves in each group were estimated by Kaplan-Meier method and assessed by Log-Rank test (p=0.05 and 0.01 %) between the control group and each treated group. Software used for statistical evaluation was SAS (SAS Institute Japan Ltd., Tokyo, Japan) and EXSUS (CAC Corp., Tokyo, Japan).

Clinical signs:

no effects observed

Description (incidence and severity):

Number of animals with abnormalities of males (3000 ppm) decreased significantly at test weeks 67-70, 76-79, 81-90 and 93 and nodule in chest of males decreased significantly at test weeks 98-100 and 102-104.

Other clinical signs were not significantly different compared with the control group.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

There was a significant increase in the mortality of the 3000 ppm female group when compared to that of the control females.
Male: 20 % (0 ppm); 18 % (1000 ppm); 14 % (3000 ppm).
Female: 18 % (0 ppm); 20 % (1000 ppm); 36 % (3000 ppm).

There were no differences in mortality (%) between the control group and each treated group in males and between the control group and the 1000 ppm group in females. However, the mortality of females in the 3000 ppm group was significantly higher than that in the control group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of males and females in the 3000ppm group and of females in the 1000ppm group were significantly lower during the treatment period.

In the 3000 ppm group, body weights of males and females were significantly lower at test weeks 2-13, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92 and 100 and at test weeks 2, 13, 28, 32, 36, 40, 44, 48, 52, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100 and 104, respectively. The ratios (%) of mean body weight to the control value (100 %) at these weeks ranged from 92 % to 97 % in males and from 86 % to 98 % in females.

In the 1000 ppm group, body weights of females were significantly lower at test weeks 8-13, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 80, 84, 96 and 100, and the ratios (%) of mean body weight to the control value (100 %) at these weeks ranged from 95 % to 98 %.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumptions of males and females in the 3000ppm group were significantly lower during the treatment period. The changes in food consumption in males in females were considered to be treatment related since they were accompanied by changes in body weight.

In the 3000 ppm group, food consumptions of males at test weeks 1, 5-8, 11-13, 16, 24, 28, 36, 40, 48, 52, 60 and 72 and the average value decreased significantly. The ratios (%) of food consumption to the control value (100 %) at these weeks ranged from 83 % to 97 % and the average value was 96%. In females, food consumptions at test weeks 1, 4-13, 16, 20, 24, 28, 32, 36, 40, 44, 48, 56, 60, 64, 68, 72, 76 and 88 and the average value decreased significantly. The ratios (%) of food consumption to the control value (100 %) at these weeks ranged from 84 % to 96 % and the average value was 92 %.

In the 1000 ppm group, food consumptions of males at test weeks 11-13, 16, 20, 24, 28, 36, 40, 48, 52, 60, 72, 80 and 84 and the average value decreased significantly. The ratios (%) of food consumption to the control value (100 %) at these weeks ranged from 93% to 96% and the average value was 97 %. However, food consumptions of males at test week 3 increased significantly and the ratio (%) of food consumption to the control value (100 %) at this week was 103 %. In females, food consumptions at test weeks 1, 6-13, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52 and 56 and the average value decreased significantly. The ratios (%) of food consumption to the control value (100 %) at these weeks ranged from 88 % to 97 % and the average value was 96 %.

The mean test substance intakes during the treatment period in the 1000 ppm and 3000 ppm groups were 41.6 mg/kg bw/day and 128.2 mg/kg bw/day in males and 50.4 mg/kg bw/day and 146.9 mg/kg bw/day in females, respectively.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the 3000 ppm group, opacity of unilateral eye of females increased significantly at test week 104 (7/32 females).
In the 1000 ppm group, opacity of unilateral eye of females increased significantly at test weeks 91, 92 (5/44 females), 95-97 (5/43 females) and 99-101 (5/42 - 6/41 females).

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

No significant changes in the 1000 ppm and 3000 ppm groups were noted compared with the control group. In the 3000 ppm group, however, WBC of 3 males and 2 females were apparently increased because of large granular lymphocytic leukemia.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In the 3000 ppm group, absolute and relative weights of the liver, spleen and kidneys increased significantly in males and females. In males, absolute and relative weights of the epididymides and relative weight of the brain increased significantly. In females, relative weights of the heart, adrenal glands and brain increased significantly and absolute weights of the heart and ovaries decreased significantly.

Significant increases of relative weights of the brain in males and females and of the heart and adrenal glands in females were considered to be related to the low body weight.

In the 1000 ppm group, absolute and relative weights of the epididymides increased significantly in males. In females, relative weight of the liver increased significantly and absolute weight of the heart decreased significantly.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In the 3000ppm group, the incidences of cyst in the mandibular lymph node and cyst in the pituitary were significantly higher than those in the control group in males. The incidences of emaciation, nodule in the chest, callus (bilateral), nodule in the pancreas, atrophy of the testis (unilateral and a total of unilateral and bilateral) and nodule in the testis (unilateral, bilateral and a total of unilateral and bilateral) were significantly lower than those in the control group. In females, the incidence of cyst in the pituitary was significantly higher than that in the control group. The incidences of gray foci of the heart and nodule in the thyroid (unilateral) were significantly lower than those in the control group.

Cyst in the pituitary in males and females with significant increased incidences was considered to be not related to the treatment with the test substance because the incidences of corresponding lesion, cyst of the pituitary, were not significantly different from those in the control group in histopathology. Emaciation, nodule in the chest, callus (bilateral) and nodule in the pancreas in males and gray foci of the heart and nodule in the thyroid (unilateral) in females with significant decreased incidences were considered to be not related to the treatment with the test substance because decreased changes of the incidence of lesions may be not regarded with toxic effects of the test substance.

Neuropathological findings:

no effects observed

Description (incidence and severity):

No indications of neurotoxicity from the limited number of examined tissues.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the 3000 ppm group, the incidences of dilatation of sinus (slight degree) in the mandibular lymph node, foci of altered cell (clear cell type) (slight degree and total number) in the liver, atrophy of seminiferous tubule (slight degree) in the testis and osseous metaplasia of sclera (slight degree) in the eye were significantly higher than those in the control group in males. The incidences of fatty change in the peripheral portion (slight degree) of the liver, microgranuloma (slight degree) in the liver, atrophy of seminiferous tubule (severe degree and total number) in the testis, hyperplasia of interstitial cell (slight degree) in the testis, decreased number of sperm (moderate and severe degree and total number) in the epididymis and diffuse hyperplasia of C cell (slight degree) in the thyroid were significantly lower than those in the control group. In females, the incidences of hyperplasia of bile duct (slight degree and total number) in the liver, chronic progressive nephrosis (moderate degree and total number) in the kidney and hyperplasia of endometrium (slight degree and total number) in the uterus were significantly higher than those in the control group. The incidences of microgranuloma (total number) in the liver, brown pigment deposit of tubular epithelium (slight degree and total number) in the kidney, osseous metaplasia of sclera (slight degree) in the eye and dilatation of duct (slight degree and total number) in the mammary gland were significantly lower than those in the control group.

Fatty change in the peripheral portion (slight degree) of the liver in males, microgranuloma (slight degree and total number) in the liver in males and females, respectively, diffuse hyperplasia of C cell (slight degree) in the thyroid in males, brown pigment deposit of tubular epithelium (slight degree and total number) in the kidney in females and dilatation of duct (slight degree and total number) in the mammary gland in females with significant decreased incidences were considered to be no toxicological significance because decreased changes of the incidence of those lesions may be not regarded with toxic effects of the test substance. The incidences of osseous metaplasia of sclera (slight degree) in the eye increased significantly in males but decreased significantly in females, being considered to be not related to the treatment with the test substance because the changes were inverse between both sexes.

In the 1000 ppm group, the incidences of atrophy of seminiferous tubule (slight degree) in the testis, decreased number of sperm (slight degree) in the epididymis and osseous metaplasia of sclera (slight degree) in the eye were significantly higher than those in the control group in males. Osseous metaplasia of sclera (slight degree) in the eye in males with a significant increased incidence was considered to be not related to the treatment with the test substance because the change was inverse in the 3000 ppm group as mentioned above. The incidence of atrophy of seminiferous tubule (severe degree) in the testis was significantly lower than that in the control group. In females, the incidence of hyperplasia of bile duct (slight degree and total number) in the liver was significantly higher than that in the control group. The incidences of fibrosis of myocardium (slight degree) in the heart and focal fatty change of cortex (slight degree) in the adrenal gland were significantly lower than those in the control group, that were considered to be not related to the treatment with the test substance because those changes were unrelated to the dose levels.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In females, the incidence of adenocarcinoma in the uterus was significantly higher than that in the control group.

In the 3000 ppm group, there were no significant increases in the incidences of neoplastic lesions in males. The incidences of interstitial cell tumor in the testis, islet cell adenoma and carcinoma in the pancreas, adenoma of anterior lobe in the pituitary and fibroma in the subcutis were significantly lower than those in the control group. In females, the incidence of adenocarcinoma in the uterus (18.8 %) was significantly higher than that in the control group (0.0 %). The incidence of C cell carcinoma in the thyroid was significantly lower than that in the control group.

In the 1000 ppm group, the incidence of fibroma in the subcutis was significantly lower than that in the control group in males. In females, no noticeable differences of the incidences in any neoplastic lesions were detected. Islet cell adenoma and carcinoma in the pancreas, adenoma of anterior lobe in the pituitary and fibroma in the subcutis in males and C cell carcinoma in the thyroid in females with significant decreased incidences were considered to be no toxicological significance because decreased incidences of neoplastic lesions is not regarded with carcinogenic effects of the test substance.

Dose descriptor:

other: NOAEL not identified

Based on:

test mat.

Sex:

male/female

Mean body weight of rats

	Males			Females
Dose level [ppm]	0	1000	3000	0	1000	3000
Body weight [g]
Week 1	111	111	111	93	93	93
Week 13	330	328	317**	186	179**	182*
Week 52	443	441	422**	228	221**	221**
Week 104	449	449	433**	299	287	261**
Week 104 Body Weight (% of Control)	-	100	96	-	96	91
Overall body weight gain [g]
Week 1 to Week 104 bw gain	338	338	322	206	194	168
0 – 104 Gain (% of control)	-	100	95	-	94	87

* p<0.05; ** p<0.01

Select incidence of microscopic neoplastic lesions in male and female rats

Site and lesion	Male			Female
No. of animals examined	50	50	50	50	50	50
Dose (ppm)	0	1000	3000	0	1000	3000
Pancreas (N)	50	50	49	50	50	50
Islet cell adenoma [Be]	10	4	1**	1	0	0
Islet cell carcinoma [Ma]	4	2	0	4	1	0
Thyroid (N)	49	50	50	50	50	50
C-cell adenoma [Be]	10	9	14	5	3	4
C-cell carcinoma [Ma]	5	2	0*	6	3	0*
Testis (N)	50	50	50	-	-	-
Interstitial cell tumor [Be]	41	40	16**	-	-	-
Uterus (N)	-	-	-	50	50	50
Adenoma [Be]	-	-	-	1	3	4
Adenocarcinoma [Ma]	-	-	-	0	5*	12**
Mammary (N)	5	4	1	50	50	50
Fibroadenoma [Be]	0	3*	1	9	8	4
Skin/subcutis (N)	50	50	50	50	50	50
Fibroma [Be]	10	3*	2*	0	0	1

N Number of animals examined at the designated site

[Be]: Benign neoplasm; [Ma]: Malignant neoplasm.

*, P<0.05; **, P<0.01 Fisher’s exact probability test

Incidence of selected histopathological findings in rat

Organ /Lesion	Male			Female
	0	1000	3000	0	1000	3000
Mandibular LN (N)	49	50	50	50	50	50
Dilation, sinus +	11	15	23*	3	2	1
Liver (N)	50	28	31	50	38	44
Hyperplasia, bile duct +	46	46	41	21	39**	43**
Hyperplasia, bile duct ++	4	4	9	1	3	6
Hyperplasia, bile duct	50	50	50	22	42**	49**
Microgranuloma +	12	10	3*	23	23	14*
Microgranuloma ++	0	0	0	8	4	2*
Microgranuloma	12	10	3*	31	27	16**
Foci, altered cell, clear cell +	2	4	10*	0	0	0
Foci, altered cell, clear cell ++	1	1	2	0	0	0
Foci, altered cell, clear cell	3	5	12*	0	0	0
Pancreas (N)	50	50	49	50	50	50
Decreased zymogen, granules, acinar cell +	4	6	2	2	5	10*
Kidney (N)	50	50	50	50	50	50
Basophilic tubular epithelium +	11	10	12	31	23	19*
Basophilic tubular epithelium ++	0	1	0	1	1	0
Basophilic tubular epithelium	11	11	12	32	24	19*
Brown pigment deposit, tubular epithelium +	13	13	14	35	28	23**
Brown pigment deposit, tubular epithelium ++	0	0	0	1	1	2
Brown pigment deposit, tubular epithelium	13	13	14	36	29	25*
Protein casts +	14	11	14	29	20	18*
Chronic progressive nephrosis +	33	29	32	11	18	12
Chronic progressive nephrosis ++	3	7	4	1	2	12**
Chronic progressive nephrosis +++	0	0	0	0	0	1
Chronic progressive nephrosis	36	36	36	12	20	25**
Testis (N)	50	50	50	-	-	-
Atrophy, seminiferous tubule +	1	12**	9**	-	-	-
Atrophy, seminiferous tubule ++	10	10	7	-	-	-
Atrophy, seminiferous tubule +++	37	23**	7**	-	-	-
Atrophy, seminiferous tubule	48	45	23**	-	-	-
Hyperplasia, interstitial cell +	41	34	11**	-	-	-
Epididymis (N)	50	50	50	-	-	-
Decreased number, sperm +	3	9	8	-	-	-
Decreased number, sperm ++	14	10	3**	-	-	-
Decreased number, sperm +++	31	26	11**	-	-	-
Decreased number, sperm	48	45	22**	-	-	-
Uterus (N)	-	-	-	50	50	50
Hyperplasia, endometrium+	-	-	-	7	10	13
Hyperplasia, endometrium++	-	-	-	0	0	1
Hyperplasia, endometrium+++	-	-	-	0	1	2
Hyperplasia, endometrium	-	-	-	7	11	16*
Cyst, endometrium +	-	-	-	23	14*	20
Eye (N)	50	50	50	50	50	50
Osseous metaplasia, sclera +	25	34	35*	8	4	1*
Mammary gland (N)	5	4	1	50	50	50
Dilatation, duct +	3	0	0	22	15	11*
Dilatation, duct ++	1	0	0	0	1	0
Dilatation, duct +++	0	0	0	1	1	0
Dilatation, duct	4	0*	0	23	17	11**

NA: Not applicable.

+: Slight. ++: Moderate. +++: Severe.

*: p<0.05); **: p<0.01

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

12.9 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

GLP and guideline compliant

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

Analysis of test substance toxicology data indicates a dopamine enhancement mode of action leading to rat uterine adenocarcinomas (see IUCLID section 7.9.3 and MOA justification document attached in IUCLID section 13). This mode of action for uterine adenocarcinoma promotion has no relevance to humans due to both qualitative and quantitative differences between rat and human. Furthermore, kinetics studies indicate that the doses where tumors were observed clearly exceeded a kinetically derived maximum tolerated dose (KMD or MTD).

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008.

An increase of adenocarcinoma in the uteri of female rats were observed at 1000 and 3000 ppm. Based on studies investigating the underlying mode of action, it can be concluded that these uterine adenocarcinoma are not of any relevance to humans. The test substance did not show a carcinogenic potential in mice.

On the basis of the available data, the test substance is not considered to be classified for carcinogenicity under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/918.

Additional information

WoE: carcinogenicity in rats 2014/8000287

The test substance was given in the feed to F344 rats (50/sex/dose) at doses of 0, 100, 300 or 1000 ppm (equal to 0, 4.4, 12.9 and 42.7 mg/kg bw/d for males and 0, 5.3, 15.5 and 50.8 mg/kg bw/d for females) for 104 weeks.

No treatment-related abnormalities were noted in mortality, general clinical observation, hematology, organ weight and body weight ratio or gross pathology in any treated groups. Body weights and food consumption in the 1000 ppm group (females) were significantly decreased for multiple weeks over the course of the study.

Adenocarcinoma of the uterus in females killed by design increased significantly in the 1000 ppm group. However, the incidences in the treatment groups were unrelated to the dose levels and no significant differences of the incidences of the tumor were noted in all females in any treatment groups. Due to the described increased incidence of spontaneous adenocarcinoma in the uterus in the F344 rat, the occurrence of adenocarcinoma in the uterus in females was considered to be unrelated to the treatment with the test substance.

A NOAEL for the study was not reported, but a NOAEL of 300 ppm (12.9 for males and 15.5 mg/kg bw/d for females) is consistent with the reported data.

WoE: carcinogenicity in rats 2014/1215781

Prior to this study, the Dose Adequacy Review Team (DART) of the Health Effects Division of the US Environmental Protection Agency reviewed the dose levels of an ongoing 2-year rat cancer study (2014/8000287) and concluded that an additional dose of 3000 ppm was required to meet the maximum tolerated dose (MTD) requirements. The agency also requested that the 1000 ppm dose be repeated to bridge the results to the ongoing rat cancer study that was being conducted at dose levels of 100, 300 and 1000 ppm.

The test substance was given in the feed to F344 rats (50/sex/dose) at doses of 0, 1000 or 3000 ppm (equal to 0, 41.6 and 128.2 mg/kg bw/d for males and 0, 50.4 and 146.9 mg/kg bw/d for females) for 104 weeks.

There was a significant increase in the mortality of the 3000 ppm female group. No treatment-related abnormalities were noted in mortality with the males. There were no treatment related clinical observations. Food consumption was decreased in all of the groups treated with the test substance. Body weights for males at 3000 ppm were statistically significantly reduced, but other treatment groups were not significantly different than control.

In the 3000 ppm group, treatment related organ weight changes included an increased absolute and relative weights of the liver, spleen and kidneys in males and females. In males, absolute and relative weights of the epididymides and relative weight of the brain increased significantly. In females, relative weights of the heart increased significantly and absolute weights of the heart and ovaries decreased significantly. At 1000 ppm, absolute and relative weights of the epididymides increased significantly in males. In females, relative weight of the liver increased significantly and absolute weight of the heart decreased significantly.

Gross histopathology of note included a decrease in the incidence of testis atrophy at 3000 ppm in the males as well as a decrease in thyroid nodules in the females. Pituitary cysts were increased in the females at 3000 ppm. Non neoplastic histopathologic findings included liver effects (increased bile duct hyperplasia and microgranuloma in all treatment groups and increased altered foci (male at 3000 ppm). In the kidney an increase in chronic progressive nephrosis (female, 3000 ppm) was noted. The testis showed decreased atrophy as well as decreased hyperplasia at 3000 ppm. In the uterus there was an increase in endometrium hyperplasia at 3000 ppm. Also at 3000 ppm, there was a decrease in the dilation of the mammary gland duct.

Neoplastic lesions present included an increased incidence of uterine adenocarcinoma at both 1000 and 3000 ppm in the females. There was a decreased incidence of testes interstitial tumors in the males at 3000 ppm as well as a decrease in thyroid C-cell carcinoma in both the males and females at 3000 ppm. Based on studies on the mode of action (see IUCLID section 7.9.3), the adenocarcinoma in the uterus are not considered to be relevant to humans.

NOAELs for all findings in this study were established in a prior rat cancer study conducted at lower doses (2014/8000287).

WoE: carcinogenicity in mice 2012/8000283

Mice were administered the test substance in the diet at doses of 0, 120, 700, 4000 (males) or 0, 120, 700, 4000/3000/2000 (females) ppm (equal to 0, 13.3, 78.7 and 445 mg/kg bw/d for males and 0, 12.9, 75.8 and 333 mg/kg bw/d for females) for 78 weeks. Dose levels for females were lowered several times because of treatment related mortality at high doses.

Treatment-related effects were restricted to the high dose level. The test substance at the dose level of 4000/3000/2000 ppm in females caused death or moribundity, low body weight, food consumption, and food efficiency, high total leukocyte, lymphocyte, and large unstained cell counts, effects on the spleen and heart and several histopathological changes including vacuolation in multiple tissues, decreases of hematopoiesis in the bone marrow, and lymphoid depletion. At the dose level of 4000 ppm in males, low body weight, and the effects on the liver (centrilobular hepatocellular hypertrophy) and submandibular gland (depletion in granular ducts) were observed. There was no increase in incidence of neoplastic lesions in any treated groups of either sex. There were no increases of rare types of tumors, earlier occurrence of spontaneous neoplasm, or other signs indicating carcinogenicity.

There were no effects at 120 or 700 ppm.

In conclusion, the test substance had no carcinogenic potential. The no-observed-adverse-effect level (NOAEL) in ICR [Crlj:CD1(ICR)] mice was considered to be 700 ppm (males, 78.7 mg/kg bw/d; females, 75.8 mg/kg bw/d) under the conditions of this study.