5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-(ethylsulfinyl)-1H-pyrazole-3-carbonitrile

Carcinogenicity

Description of key information

Oral:
NOAEL (toxicity, chronic, 104 weeks, rat) = 0.85 (males) and 1.17 (females) mg/kg bw/day

NOAEL (pre-neoplastic lesions, chronic, 104 weeks, rat) = 3.21 (males) and 4.40 (females) mg/kg bw/day

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:

key study

Study period:

In-life dates: 15 January 1998 - 31 January 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Well-documented study profile, but no original study report available.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.4300 (Combined Chronic Toxicity / Carcinogenicity)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

Guideline in place during study conduct: adopted in 1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

Current version adopted in 2018

Deviations:

no

GLP compliance:

yes

Species:

rat

Strain:

other: Wistar rats R.J: WHOPS AF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: R. Janvier, Le Genest St Isle, France
- Age at study initiation: 6 weeks
- Weight at study initiation: 246.0-249.4 g (males), 178.0-182.7 g (females)
- Housing: rats were housed in groups of 5/sex/cage at the start of the study. Cages were suspended over trays.
- Diet: Ground diet, “M20 contrôlé” (Pietremont BP 59, Provins Cedex, France), ad libitum
- Water: filtered and softened water from the municipal water supply, ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 15 Jan 1998 To: 31 Jan 2000

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): approximately every 4 weeks
- Mixing appropriate amounts with (Type of food): Ground diet, “M20 contrôlé” (Pietremont BP 59, Provins Cedex, France). The test substance was ground to a fine powder before being incorporated into the diet by dry mixing.
- Storage temperature of food: at -18 °C

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYTICAL STUDY: the stability of the test substance in the diet was demonstrated prior to the start of the study in the previous subchronic rat study (study Number SA 97233). Analysis of the diets was performed using High Performance Liquid Chromatography (HPLC) on a C18 column and ultraviolet (UV) detection at 277 nm. During the current study, the homogeneity of the test substance in diet was verified on the first formulation to demonstrate adequate formulation procedures at 5, 20, 75 and 250 ppm. In addition, the concentration of each diet preparation was verified prior to administration to the animals for the first three formulations, and then every 3 months thereafter.
- Stability: in the previously conducted study, samples of 5 and 2500 ppm diets were found to be stable after storage at room temperature for up to 7 weeks, or after frozen storage for up to 45 days followed by 7 days at room temperature.
- Homogeneity analysis: all the results for homogeneity checks were within the target range of 85-115% of the nominal concentration, except for one result out of nine at 5 ppm which was slightly below the target ranges (80%).
- Concentration analysis: all results for concentration checks were within 85-114% of the nominal concentration. One result for the ninth formulation at 5 ppm was slightly below the target ranges (84%).

Duration of treatment / exposure:

52 weeks (chronic phase)
52 weeks and 13 weeks post-exposure observation period (recovery/reversibility phase)
104 weeks (carcinogenicity phase)

Frequency of treatment:

daily, 7 days/week

Post exposure period:

13 weeks (after chronic exposure for 52 weeks)

Dose / conc.:

5 ppm (nominal)

Remarks:

actual test substance intake: males: 0.25 mg/kg bw/day, females: 0.34 mg/kg bw/day
as calculated from the reported body weight and food intake values during Weeks 1-48 (chronic phase)

Dose / conc.:

5 ppm (nominal)

Remarks:

actual test substance intake: males: 0.22 mg/kg bw/day, females: 0.29 mg/kg bw/day
as calculated from the reported body weight and food intake values during Weeks 1-101 (carcinogenicity phase)

Dose / conc.:

20 ppm (nominal)

Remarks:

actual test substance intake: males: 0.98 mg/kg bw/day, females: 1.34 mg/kg bw/day
as calculated from the reported body weight and food intake values during Weeks 1-48 (chronic phase)

Dose / conc.:

20 ppm (nominal)

Remarks:

actual test substance intake: males: 0.85 mg/kg bw/day, females: 1.17 mg/kg bw/day
as calculated from the reported body weight and food intake values during Weeks 1-101 (carcinogenicity phase)

Dose / conc.:

75 ppm (nominal)

Remarks:

actual test substance intake: males: 3.73 mg/kg bw/day, females: 5.01 mg/kg bw/day
as calculated from the reported body weight and food intake values during Weeks 1-48 (chronic phase)

Dose / conc.:

75 ppm (nominal)

Remarks:

actual test substance intake: males: 3.21 mg/kg bw/day, females: 4.40 mg/kg bw/day
as calculated from the reported body weight and food intake values during Weeks 1-101 (carcinogenicity phase)

Dose / conc.:

250 ppm (nominal)

Remarks:

actual test substance intake: males: 12.39 mg/kg bw/day, females: 16.51 mg/kg bw/day
as calculated from the reported body weight and food intake values during Weeks 1-48 (chronic phase)

Dose / conc.:

250 ppm (nominal)

Remarks:

actual test substance intake: males: 10.79 mg/kg bw/day, females: 14.68 mg/kg bw/day
as calculated from the reported body weight and food intake values during Weeks 1-101 (carcinogenicity phase)

No. of animals per sex per dose:

10 (chronic phase)
15 (satellite control and high dose group of the recovery/reversibility phase)
60 (carcinogenicity phase)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: the dose levels were selected based on the results of a previously conducted 90-day rat toxicity study (study Number SA 97233), in which rats were fed with 5, 20, 500 and 2500 ppm test substance in the diet. Treatment-related mortalities and adverse effects on liver and thyroid gland were observed at dose levels of 500 and 2500 ppm. The NOAEL for rats from this study was set at 20 ppm. Therefore, dose levels of 5, 20, 75 and 250 ppm were selected for the present study.
- Rationale for selecting satellite groups: satellite groups were selected to study recovery/reversibility of effects.
- Post-exposure recovery period in satellite groups: 13 weeks

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were checked for clinical signs, moribundity and mortality twice daily (once daily on weekends or public holidays). Observed clinical signs were recorded at least once daily for all animals. The nature, onset, severity, duration and recovery of clinical signs were recorded. Cages and cage-trays were inspected daily for evidence of ill-health such as blood or loose faeces.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: during acclimatisation, detailed physical examination of the animals was conducted. During the study, detailed physical examinations including palpation for masses were performed twice monthly during the first 13 weeks of the study and weekly thereafter. The onset, location and dimension of the masses were recorded. Debilitated animals were observed carefully and were eventually isolated.

BODY WEIGHT: Yes
- Time schedule for examinations: each animal was weighed twice during the acclimatisation period, then weekly for the first 13 weeks of study and every 4 weeks thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined: Yes, weekly during the first 13 weeks of treatment, and every 4 weeks thereafter.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes, for each week and for Weeks 1-48 (chronic phase) and for Weeks 1-101 (carcinogenicity phase). Compound intake was calculated using the average of the body weight at the beginning of the interval and the body weight at the end of the interval for the measurement period for weeks 1 through 13, and was calculated using the single body weight available for the interval for the measurement period for weeks 17 through 101.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: funduscopic (indirect ophthalmoscopy) and biomicroscopic (slit lamp) examinations were performed during the acclimatisation phase and on all surviving animals at one and two years. Each eye was firstly examined by direct ophthalmoscopy, and then after instillation of an atropinic agent (Mydriaticum, Merck Sharp and Dohme), each eye was re-examined by means of a slit lamp and an indirect ophthalmoscope.
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during Months 6 and 12 on the surviving animals of the interim sacrifice groups (chronic phase) and on the first ten surviving animals of the recovery groups and of the terminal sacrifice groups (carcinogenicity phase). Haematology was also performed during Month 15 on the first ten surviving animals of the recovery groups and during Months 18 and 24 on the first ten surviving animals of the terminal sacrifice groups (carcinogenicity phase).
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes, overnight
- How many animals: all surviving animals of the chronic phase, the first 10 surviving animals of the recovery phase and carcinogenicity phase groups, respectively
- Parameters checked: haematocrit (HCT), haemoglobin (HGB), leukocyte count (WBC), erythrocyte count (RBC), platelet count, blood clotting measurements (prothrombin time), leukocyte differential count, mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during Months 6 and 12 on the surviving animals of the interim sacrifice groups (chronic phase) and on the first ten surviving animals of the recovery groups and of the terminal sacrifice groups (carcinogenicity phase). Clinical chemistry measurements were was also performed during Month 15 on the first ten surviving animals of the recovery groups and during Months 18 and 24 on the first ten surviving animals of the terminal sacrifice groups (carcinogenicity phase).
- Animals fasted: Yes, overnight
- How many animals: all surviving animals of the chronic phase, the first 10 surviving animals of the recovery phase and carcinogenicity phase groups, respectively
- Parameters checked: calcium, chloride, phosphorus, potassium, sodium, albumin, creatinine, urea nitrogen, total cholesterol, glucose (fasting), total bilirubin, total protein (TP), triglycerides, alkaline phosphatase (ALK), alanine aminotransferase (ALT/SGPT), aspartate aminotransferase (AST/SGOT)

URINALYSIS: Yes
- Time schedule for collection of urine: during Months 6 and 12 on the surviving animals of the interim sacrifice groups (chronic phase) and on the first ten surviving animals of the recovery groups and of the terminal sacrifice groups (carcinogenicity phase). Urinalysis was also performed during Month 15 on the first ten surviving animals of the recovery groups and during Months 18 and 24 on the first ten surviving animals of the terminal sacrifice groups (carcinogenicity phase).
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes, overnight
- Parameters checked: appearance, volume, refractive index, pH, sediment (microscopic), protein, glucose, ketones, bilirubin, blood/ red blood cells, urobilinogen

NEUROBEHAVIOURAL EXAMINATION: No, since separate neurotoxicity studies are available for the test substance.

OTHER: THYROID HORMONE ANALYSIS
- Time schedule for collection of blood: at Weeks 2, 9, 24, 52 and at Weeks 2, 6 and 13 of the recovery phase
- Animals fasted: Yes
- How many animals: all surviving animals of the chronic and recovery groups
- Parameters checked: thyrotropin stimulating hormone (TSH), triiodothyronine (T3) and thyroxine (T4)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Table 1 under “Any other information on materials and methods incl. tables)
HISTOPATHOLOGY: Yes, (see Table 1 under “Any other information on materials and methods incl. tables)
All animals that died and those sacrificed on schedule were subjected to gross pathological examination. Organ weights of liver, heart, spleen, kidney, testes, epididymides, ovaries, uterus, brain, adrenal gland were determined. Paired organs were weighed together. During the chronic phase, histopathological examinations were performed for all animals in the control and high dose groups for all collected tissues. In addition, liver, lung, kidney, thyroid glands (with parathyroids), and tumours were examined for all the animals from the intermediate dose groups. During the reversibility phase, histopathological examinations were performed on target organs (liver and thyroid glands) as identified in the chronic phase of the study. All tissues were examined in animals which died prematurely (before scheduled sacrifice). In addition, all suspected tumours and gross findings detected at necropsy were also examined in all animals. During carcinogenicity phase, histopathological examinations were performed in all animals from all groups.

Statistics:

Mean values and standard deviations (SD) were calculated for each sex separately for each group at each time period.

Variables analysed:
- mortality
- body weights
- body weight changes
- food consumption
- haematology parameters (except eosinophils, basophils, monocytes and large unstained cells)
- clinical chemistry parameters
- urinary parameters (only pH, volume and refractive index)
- organ weight parameters
- organ/body weight ratios
- organ/brainweight ratios
- hormone data
- tumour incidence

A combination of standard ANOVA and non parametric data analysis techniques was used. Detailed descriptions of the methods for statistical analysis are given under “Any other information on materials and methods incl. tables”.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment-related clinical signs noted at any dose level during any phase of the study.

Mortality:

no mortality observed

Description (incidence):

No treatment-related effect on mortality was noted at any dose level for either sex.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Weeks 0-48: statistically signficantly increased body weight gain in males at 20, 75 and 250 ppm and females at 75 and 250 ppm compared to controls

In males treated at 20, 75 and 250 ppm and in females treated at 75 and 250 ppm, body weight gain was statistically significantly increased from Week 0 to Week 48, when compared to controls. In females treated at 20 ppm, body weight gain was statistically significantly increased for the first 4 weeks of the study. From Week 48 to Week 101, no significant differences in body weight gain were recorded in both males and females, when compared to controls. During the recovery period, body weight gain in animals of both sexes treated at the high dose level was not significantly different compared to controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

all dose levels (males): higher overall mean food consumption compared to controls; 5, 75 and 250 ppm (females): lower overall mean food consumption females compared to controls

The overall mean food consumption was higher in males at all dose levels, when compared to controls, which was consistent with the observed increase in body weight gain in males. Generally, the overall mean food consumption was lower in females at 5, 75 and 250 ppm. The differences achieved statistical significance on several occasions but no dose-related effect was noted.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related changes at the ophthalmological examination at one and two years.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

75 and 250 ppm (stat. significant): prothrombin time increased in males and decreased in females (reversible during recovery); 250 ppm (stat. significant): higher platelet counts at Months 12 and 24 in females

Prothrombin times were higher in males and lower in females throughout the study at 75 and 250 ppm. After the recovery period, no relevant differences were noted between controls and the high dose group indicating that these effects were reversible. In addition at 250 ppm in females, the mean platelet counts were statistically significantly higher on Months 12 (+25%, α=0.001) and 24 (+35%, α=0.01).
Some other slight variations in erythrocyte parameters noted at 250 ppm were as follows. In the high dose males, lower mean haemoglobin (-2%, α=0.05) and mean corpuscular haemoglobin concentration (-1%, α=0.05), were observed at Month 12. In the 250 ppm females, higher mean red blood cell count (+4%, α=0.01) was noted at Month 6, and lower mean corpuscular volume and mean corpuscular haemoglobin (by about 5% and 6%, respectively) were observed at Months 6, 12 and 24. However, these changes were of low magnitude and high inter-individual variability was noted. Therefore, they were not considered toxicologically significant.
Other statistically significant changes found in the haematological parameters were considered to be incidental and unrelated to treatment as they were not dose-related and/or not biologically relevant.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

250 ppm: increased total cholesterol concentration (f), increased protein concentration (m, f); 75 and 250 ppm (m, f): decreased bilirubin (reversible during recovery)

In females at 250 ppm, mean total cholesterol concentration was higher at Months 6 (+44%, α=0.001), 12 (+55%, α=0.001), 18 (+84%, α=0.01) and 24 (+33%, not statistically significant). Higher mean total protein concentrations were observed at 250 ppm in males and females in Months 6 (approx. +10%, α=0.001), 12 (approx. +7%, α=0.001), and in females only on Month 18 (+7%, α=0.05).
Mean total bilirubin concentrations were lower at 75 and 250 ppm in both sexes on Months 6, 12, 18 and 24, and at 20 ppm in males on Month 18. The percentages of changes (compared to controls) are presented in Table 2 under “Any other information of results incl. tables”. After the 3-month recovery period, no relevant changes were noted between controls and the high dose group indicating that the effects were reversible.
Also at 75 and 250 ppm, a few statistically significant changes were noted in enzyme activities of ASAT, ALAT and AP in males and/or females at different sampling times. However, these changes were of low magnitude and not consistent over time and thus were not considered to be toxicologically significant. Other statistically significant variations noted in clinical chemistry were of low magnitude or not dose-related, thus they were not considered to be toxicologically significant.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

urinary pH: increased in females at 250 ppm at Months 6 and 12 and at 75 ppm at Month 12 (reversible during recovery); refractive index: slightly decreased in females at 250 ppm at Month 6 (reversible during recovery)

Urinary pH values were higher in females at 250 ppm at Months 6 and 12 (+9% and +7%, respectively, α=0.001) and at 75 ppm at Month 12 (+7%, α=0.05). In addition, for females at 250 ppm the mean refractive index was slightly lower at Month 6. No treatment-related changes were noted at Months 18 and 24 in either males or females. After a 3-month recovery period, no relevant changes were noted between controls and the high dose group indicating that these findings were reversible. Thus, no meaningful toxicological changes in urinalysis were observed in the study.

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):

75 and/or 250 ppm (m, f): increase in absolute and/or relative weights of liver and thyroid

- Chronic phase: treatment-related organ weight changes were noted in the liver and thyroid gland (Table 3 under “Any other information of results incl. tables”), which were accompanied by non-neoplastic histopathological changes (see section “HISTOPATHOLOGY: NON-NEOPLASTIC” below). There was a statistically significant, dose-related, higher mean liver weight at 75 ppm in females only and at 250 ppm in both sexes. The mean thyroid weight was increased (statistically significant) at 75 ppm in males only and at 250 ppm in both sexes.

- Reversibility phase: no treatment-related changes in organ weights were noted after the 3-month recovery period.

- Carcinogenicity phase: at the end of the carcinogenicity phase, treatment-related organ weight changes were noted in the liver and thyroid gland (see Table 3 under “Any other information of results incl. tables”), which were accompanied by either non-neoplastic or neoplastic histopathological changes (see sections “HISTOPATHOLOGY: NON-NEOPLASTIC” and “HISTOPATHOLOGY: NEOPLASTIC” below). Mean liver weights were increased (statistically significant) at 75 ppm in females and 250 ppm in both sexes. There was a statistically significant higher mean thyroid weight at 75 and 250 ppm in both males and females. In males, this change was only statistically significant when expressed as the ratio to brain weight. A few statistically significant minimal differences were observed as compared to control group, namely a higher mean kidney weight at 250 ppm in both sexes, at 75 ppm in females, and at 5 ppm in males; and a higher mean heart weight at 250 ppm in females. These differences, only noted at the end of the 104-week phase of the study, were minimal (8-13%), not observed for all the modes of expression of the organ weights, not dose-related, and not correlated to gross or microscopic changes. Therefore, they were not considered to be biologically or toxicologically significant.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related gross pathologic changes were noted at any dose level during any phase of the study.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

75 ppm (f): histopathological alterations in thyroid; 250 ppm (m, f): histopathological alterations in thyroid and liver

- Chronic phase: treatment-related changes were noted in the liver and thyroid gland as follows. In the liver, centrilobular hepatocyte hypertrophy attributable to treatment was noted in all females at 250 ppm. This change characterized by an appreciable increase in the size of hepatocytes in the area of the central vein was generally graded slight to mild and is considered to represent a minor adaptive change. In the thyroid, follicular cell hypertrophy was noted in animals of both sexes dosed at 250, 75 or 20 ppm. This change which is characterized by an increase in height of the follicular epithelial cells accompanied by a decrease in the follicular size and in colloid content was graded slight to moderate. There was evidence of a dose-effect relationship in both sexes, more prominently in females than in males. In addition, colloid mineralisation was observed in a small proportion of males (2/9) and females (4/10) at 250 ppm.

- Reversibility phase: in the liver, there was a markedly lower occurrence of periportal hepatocyte microvacuolation in females at 250 ppm (2/11) compared to the control (9/15). Centrilobular hepatocyte hypertrophy, which was considered to be treatment-related at the end of the 52-week chronic phase of the study in females at 250 ppm, was fully reversible. In the thyroid gland, the occurrence of colloid mineralization, characterised by the presence of basophilic material within the colloid of the majority of the follicles, was higher in males (7/14) and females (4/11) from the 250 ppm group as compared to controls (0/12 and 0/15 for males and females, respectively). Colloid mineralisation in the thyroid gland was also observed in 1 of the 4 high-dose females which died prior to the scheduled sacrifice. This was the only treatment-related finding noted in the animals which died early for this phase of the study. In addition in the thyroid, the occurrence of slight follicular cell hypertrophy was still higher in males from the 250 ppm group (5/14) as compared to the control group (1/13), indicating that reversibility was in progress but incomplete in males. However, in females which were more affected than males at the end of the 52-week chronic phase, this change was fully reversible.

- Carcinogenicity phase: in the liver, bile duct hyperplasia and sclerosis and focal sinusoidal dilatation were noted in many animals with the incidence and severity being slightly higher in females at 250 ppm. Also in the liver, eosinophilic and basophilic tigroid foci of cellular alteration were noted in many animals from all groups. The incidence of both types (and the severity of tigroid foci) was slightly higher in males dosed at 250 ppm in comparison with controls. In females, the incidence of tigroid altered hepatocyte foci was lower in animals dosed at 250 ppm with no effect on the incidence of eosinophilic foci of cellular alteration. In the thyroid gland, colloid mineralisation was the predominant finding in rats of both sexes at 250 ppm and females at 75 ppm. Females at 250 ppm also showed significant diffuse follicular hypertrophy. These changes are consistent with minor, chronic stimulation. In the kidneys, chronic progressive nephropathy was noted in most animals from all groups. The severity, but not the incidence, was slightly higher in rats of both sexes at 250 ppm. In addition, the incidence of arteritis/periarteritis in the kidney was marginally higher than controls in females at 250 ppm. This change may be related to the increased severity of chronic progressive nephropathy. In the lungs, foci of alveolar macrophages were noted in many animals from all groups. The incidence was marginally higher than controls in females dosed at 250 ppm.
Selected neoplastic microscopic findings from the carcinogenicity phase of the study are presented in Table 4 under “Any other information of results incl. tables”.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

250 ppm: slight, but not statistically significant increase in follicular hyperplasia and adenomas in the thyroids (m, f) and hepatocellular adenomas (m)

Selected neoplastic microscopic findings from the carcinogenicity phase of the study are presented in Table 5 under “Any other information of results incl. tables”.
- Reversibility and chronic phases: there was no evidence of treatment-related increased incidence of proliferative changes at the end of either the chronic or reversibility/recovery phases of the study.

- Carcinogenicity phase: for the unscheduled deaths, there was no indication of a significantly higher incidence of tumors overall in either sex. Findings in the target organs (liver and thyroid) in the animals that died prematurely are as follows. Thyroid follicular cell adenoma was seen in four males dosed at 250 ppm with no similar lesion in controls. There was no evidence of an effect of treatment in females. Focal follicular cell hyperplasia was seen in one control male, one 5 ppm male, four 250 ppm males and one 250 ppm female. In the liver, hepatocellular adenoma was seen in one male dosed at 250 ppm. At the scheduled sacrifice, there was no evidence of an effect of treatment in the thyroid. Follicular cell adenoma was noted in two females in the 250 ppm group. In the liver, hepatocellular adenoma was noted in two males at 250 ppm and in one female at 75 ppm. For the overall incidence (scheduled + unscheduled deaths), when focal follicular cell hyperplasia is included in the analysis, a higher incidence of focal proliferative lesions in comparison with controls becomes apparent, especially in males (see Table 4 under “Any other information of results incl. tables”). In the context of the stimulatory effect of treatment on the thyroid gland, these results provide evidence of a treatment-related effect on focal proliferative lesions at 250 ppm especially in males. In the liver, hepatocellular adenoma was found in three males at 250 ppm and in one female at 75 ppm with no similar lesion in controls. There was no indication of a significantly higher overall tumor incidence in either sex. Although a slight increase was observed at 250 ppm for follicular hyperplasia and adenomas in the thyroids (both sexes) and for adenomas in the liver (males only), no statistical differences were noted. A variety of spontaneous tumors was noted in animals from all groups with no evidence of an effect of treatment. Tumors were commonly encountered (10 or more in a group) in the pituitary gland, thyroid gland (C-cells), subcutaneous tissue (males) and mammary gland (females). The spectrum was consistent with the range of spontaneous lesions encountered in aging rats of this strain.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

THYROID HORMONE ANALYSIS
The test substance induced an imbalance of thyroid hormones resulting in higher levels of TSH and lower levels of T4 in females at 20, 75 and 250 ppm and in males at 75 and 250 ppm. These changes were relatively consistent throughout the study for females at 75 and 250 ppm and for males at 250 ppm but appeared more sporadically and were not statistically significant in females at 20 ppm and in males at 75 ppm. No dose-related or consistent changes in T3 were noted for any group. During the recovery period, TSH and T4 returned to control levels within 2 weeks of the withdrawal of treatment at 250 ppm. The imbalance of TSH and T4 was most likely secondary to a treatment-induced increase in the metabolic activity of the liver causing an increased biliary clearance of T4 and consequently, an over stimulation of the thyroid due to increased levels of TSH. Consistent with this mechanism, an increase in liver and thyroid weights were observed at the sacrifice intervals for the chronic (after 52 weeks) and carcinogenicity (after 104 weeks) phases in both sexes at 250 ppm. In addition, liver weight was increased in females at 75 ppm for both of these sacrifice intervals while thyroid weight was increased at 75 ppm in males only after 52 weeks and in both sexes at 75 ppm after 104 weeks.

Dose descriptor:

NOAEL

Remarks:

Toxicity (chronic phase)

Effect level:

20 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects; no changes in liver and thyroid weights and no toxicologically relevant changes in histopathology

Remarks on result:

other: actual test substance intake: males: 0.98 mg/kg bw/day, females: 1.34 mg/kg bw/day as calculated from the reported body weight and food intake values during Weeks 1-48 (chronic phase)

Dose descriptor:

LOAEL

Remarks:

Toxicity (chronic phase)

Effect level:

75 ppm (nominal)

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

other: see "Remark"

Remarks on result:

other: actual test substance intake: males: 3.73 mg/kg bw/day, females: 5.01 mg/kg bw/day as calculated from the reported body weight and food intake values during Weeks 1-48 (chronic phase)

Key result

Dose descriptor:

NOAEL

Remarks:

Toxicity (carcinogenicity phase)

Effect level:

20 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects; no changes in liver and thyroid weights and no toxicologically relevant changes in histopathology

Remarks on result:

other: actual test substance intake: males: 0.85 mg/kg bw/day, females: 1.17 mg/kg bw/day as calculated from the reported body weight and food intake values during Weeks 1-101 (carcinogenicity phase)

Key result

Dose descriptor:

LOAEL

Remarks:

Toxicity (carcinogenicity phase)

Effect level:

75 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

other: see 'Remark'

Remarks on result:

other: actual test substance intake: males: 3.21 mg/kg bw/day, females: 4.40 mg/kg bw/day as calculated from the reported body weight and food intake values during Weeks 1-101 (carcinogenicity phase)

Key result

Dose descriptor:

NOAEL

Remarks:

Pre-neoplastic lesions

Effect level:

75 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: up to a dose level of 75 ppm, no increase in the incidence of tumours was observed between treated and control animals

Remarks on result:

other: actual test substance intake: males: 3.21 mg/kg bw/day, females: 4.40 mg/kg bw/day as calculated from the reported body weight and food intake values during Weeks 1-104 (carcinogenicity phase)

Key result

Dose descriptor:

LOAEL

Remarks:

Pre-neoplastic lesions

Effect level:

250 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

Remarks on result:

other: actual test substance intake: males: 10.79 mg/kg bw/day, females: 14.68 mg/kg bw/day as calculated from the reported body weight and food intake values during Weeks 1-104 (carcinogenicity phase)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

75 ppm

System:

other: hepatobiliary and endocrine system

Organ:

liver

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Table 2. Percent change in bilirubin relative to control at 20, 75 and 250 ppm

Parameter	Interval	Gender	Dose level (ppm in diet)
			20	75	250
Total Bilirubin Concentrations (TBIL)	Month 6	Males	NC	-24%	-39%
		Females	NC	-44%	-57%
	Month 12	Males	NC	-25%	-42%
		Females	NC	-35%	-60%
	Month 18	Males	-31%	-36%	-50%
		Females	NC	-33%	-47%
	Month 24	Males	NC	-30%	-46%
		Females	NC	-41%	-50%

NC = no relevant changes

Table 3. Organ weights: Percent change relative to controls in liver and thyroid weights at 5, 20, 75 and 250 ppm

Organ	Parameter	Dose level (ppm in diet)
		5	20	75	250
Males
Chronic Phase
Liver	Absolute weight	NC	NC	NC	+30 %
	Ratio to body weight	NC	NC	NC	+ 19%
	Ratio to brain weight	NC	NC	NC	+31%
Carcinogenicity Phase
Liver	Absolute weight	NC	NC	NC	+23 %
	Ratio to body weight	NC	NC	NC	+25%
	Ratio to brain weight	NC	NC	NC	+25%
Females
Chronic Phase
Liver	Absolute weight	NC	NC	+ 12%	+45%
	Ratio to body weight	NC	NC	+ 14%	+46%
	Ratio to brain weight	NC	NC	+ 18%	+48%
Carcinogenicity Phase
Liver	Absolute weight	NC	NC	+ 13%	+38%
	Ratio to body weight	NC	NC	+ 14%	+37%
	Ratio to brain weight	NC	NC	+ 14%	+38%
Males
Chronic Phase
Thyroid	Absolute weight	NC	NC	+36%	+68%
	Ratio to body weight	NC	NC	+21%	+50%
	Ratio to brain weight	NC	NC	+33%	+70%
Carcinogenicity Phase
Thyroid	Absolute weight	NC	NC	+39%	+21%
	Ratio to body weight	NC	NC	+38%	+21%
	Ratio to brain weight	NC	NC	+36%	+21%
Females
Chronic Phase
Thyroid	Absolute weight	NC	NC	NC	+20%
	Ratio to body weight	NC	NC	NC	+24%
	Ratio to brain weight	NC	NC	NC	+27%
Carcinogenicity Phase
Thyroid	Absolute weight	NC	NC	+28%	+36%
	Ratio to body weight	NC	NC	+33%	+35%
	Ratio to brain weight	NC	NC	+30%	+34%

Table 4. Non-Neoplastic Microscopic Findings in Rats at Week 104 (Carcinogenicity Phase)

Observation		Males					Females
		Dose Level (ppm in diet)					Dose Level (ppm in diet)
		0	5	20	75	250	0	5	20	75	250
LIVER	Number examined	60	60	60	60	60	60	60	60	60	60
	Hyperplasia, Bile Ducts:
	Slight	23	33	29	26	30	16	22	15	24	18
	Mild	14	1	4	14	8	3	1	2	5	14
	Moderate	3	0	1	0	1	0	0	0	0	1
	Total	40	34	34	40	39	19	23	17	29	33
	Sclerosis, Bile Ducts:
	Slight	18	29	26	28	33	9	13	6	17	22
	Mild	11	9	10	11	9	1	0	0	0	7
	Moderate	0	0	0	0	0	0	0	1	0	0
	Total	29	38	36	39	42	10	13	7	17	29
	Dilatation, Sinusoidal, Focal:
	Slight	4	2	9	8	5	14	13	14	17	28
	Mild	0	0	0	0	0	1	0	0	0	2
	Total	4	2	9	8	5	15	13	14	17	30
	Cellular Alteration, Basophilic, Tigroid, Focal:
	Slight	30	33	38	33	37	30	30	27	32	17
	Mild	2	2	3	6	10	10	13	8	4	4
	Moderate	0	0	0	0	3	2	0	0	2	0
	Marked	0	0	0	0	1	0	0	0	1	0
	Total	32	35	41	39	51	42	43	35	39	21
	Cellular Alteration, Eosinophilic Type, Focal:
	Slight	23	20	16	16	32	22	32	24	17	29
	Mild	3	3	3	4	8	5	3	6	6	2
	Moderate	0	0	1	0	0	0	0	2	0	0
	Total	26	23	20	20	40	27	35	32	23	31
THYROID	Number examined	60	60	59	60	59	59	59	60	60	60
	Mineralization, Colloid:
	Slight	4	4	4	9	12	0	1	3	5	16
	Mild	3	2	0	2	6	0	0	0	2	16
	Moderate	1	1	1	3	11	0	0	0	2	10
	Marked	1	0	0	0	0	0	0	0	0	0
	Total	9	7	5	14	29	0	1	3	9	42
	Hypertrophy, Follicular, Diffuse:
	Slight	1	0	2	3	3	0	0	0	3	14
	Mild	1	1	2	0	0	0	0	0	1	6
	Total	2	1	4	3	3	0	0	0	4	20

Table 5. Neoplastic microscopic findings in thyroids of rats from carcinogenicity phase (Week 104)

Organ	Pre-neoplastic lesions	Dose level (ppm in diet)
		0	5	20	75	250
Malesat terminal sacrifice (Week 104)
THYROID	Number examined	60	60	59	60	59
	Hyperplasia, follicular cell, focal	2	1	0	1	5
	Adenoma, follicular cell	0	0	0	0	4
	Carcinoma, follicular cell	0	0	0	0	0
	Total with focal proliferative lesions	2	1	0	1	9
Femalesat terminal sacrifice (Week 104)
THYROID	Number examined	59	59	60	60	60
	Hyperplasia, follicular cell, focal	0	1	0	1	2
	Adenoma, follicular cell	0	0	0	0	2
	Carcinoma, follicular cell	0	0	1	1	0
	Total with focal proliferative lesions	0	1	1	2	4

Conclusions:

Chronic dietary administration of the test substance to rats resulted in a slight, but not statistically significant increased incidence of thyroid adenomas (both sexes) and liver adenomas (males only) observed at 250 ppm. The lesions in the thyroid were consistent with a sequence of microscopic changes which frequently result from chronic hormonal stimulation of this organ in rats, thereby indicating only low human relevance. There was no indication of a significantly higher overall tumour incidence in either sex. A variety of spontaneous tumours was noted in animals from all groups with no evidence of an effect of treatment. Tumours were commonly encountered (10 or more in a group) in the pituitary gland, thyroid gland (C-cells), subcutaneous tissue (males) and mammary gland (females). The spectrum was consistent with the range of spontaneous lesions encountered in aging rats of this strain. Overall, due to missing human relevance and /or limited toxicological significance as determined by missing statistical significance, the observed pre-neoplastic findings are not considered as conclusive for classification purposes.
The No Observed Adverse Effect level (NOAEL) with regard to pre-neoplastic lesions was 75 ppm corresponding to 3.21 and 4.40 mg/kg bw/day in males and females, respectively.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

0.85 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 1 and 2) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

System:

other: hepatobiliary and endocrine system

Organ:

liver

thyroid gland

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Please refer to the information provided in IUCLID section 13.2.

Justification for classification or non-classification

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

Additional information

The long-term and carcinogenic effects of the substance were investigated in male and female Wistar (R.J: WHOPS AF) rats in a combined study of chronic toxicity for 52 weeks and carcinogenicity for 104 weeks according to guidelines OECD 453 and EPA OPPTS 870.4300 (M-203024-02-1, 2002). Sixty animals per sex and group were administered the test substance daily for 7 days/week at dietary levels of 0, 5, 20, 75, and 250 ppm during the chronic and carcinogenicity exposure. For the carcinogenicity study, the dietary dose levels corresponded to a mean achieved compound intake of 0.22, 0.85, 3.21 and 10.79 mg/kg bw/day in males and 0.29, 1.17, 4.40 and 14.68 mg/kg bw/day in females, respectively (as calculated from the reported body weight and food intake values during Weeks 1-101). In addition, satellite groups of each 10 animals per sex for control and high dose treatment were included in the chronic study to investigate the reversibility of effects after a 13-week recovery period.

During all study phases (chronic study with or without recovery and carcinogenicity study), no treatment-related mortalities and no clinical signs were observed. The body weight evolution of males and females at 75 and 250 ppm was slightly superior to controls especially during the first year of treatment. The overall food consumption was generally higher in males and lower in females.

Haematological examination revealed that prothrombin time was higher in males and lower in females throughout the study at dose levels of 75 and 250 ppm, respectively. During the 13-week recovery phase, no relevant differences were noted in prothrombin times between controls and the high dose group, indicating that these effects were reversible. At 250 ppm, the mean platelet counts were statistically significantly higher in females at Months 12 and 24. All other changes in haematological parameters at 250 ppm (mean haemoglobin, mean corpuscular haemoglobin concentration and red blood cell counts) were of low magnitude and high inter-individual variability, and were therefore not considered to be toxicologically significant.

At clinical chemistry analysis, higher mean cholesterol levels were noted in females throughout the chronic and carcinogenicity phase. Higher mean total protein concentrations were observed at 250 ppm in males and females at Months 6 and 12, and in females only at Month 18. In both sexes, a decrease in total bilirubin was observed at 250 ppm at several intervals during the study. All of these changes were found to be completely reversible in animals of the at 250 ppm satellite groups during the 13-week recovery period after 52 weeks of continuous treatment. A few statistically significant changes were noted in enzyme activities of ASAT, ALAT and AP in males and/or females at different sampling times, but these changes were not regarded toxicologically relevant, since they were of low magnitude and not consistent. No toxicologically relevant changes in urinary parameters were observed during the whole study period.

The analysis of thyroid hormones revealed higher levels of TSH and lower levels of T4 in females treated with 20, 75 and 250 ppm and in males treated with 75 and 250 ppm. These changes were relatively consistent throughout the study for females at 75 and 250 ppm and for males at 250 ppm, but appeared more sporadically and were not statistically significant in females at 20 ppm and in males at 75 ppm. No dose-related or consistent changes in T3 hormone were noted for any group. During the 13-week recovery period, TSH and T4 returned to control levels within 2 weeks of the withdrawal of treatment at 250 ppm.

At necropsy, no macroscopic lesions were observed in any treatment group. An increase in liver and thyroid weights was observed at the sacrifice intervals for the chronic (after 52 weeks) and carcinogenicity (after 104 weeks) phases in both sexes at 250 ppm. The increase in thyroid weight was correlated with the elevated levels of TSH, causing an over-stimulation of this organ. In addition, liver weight was increased in females at 75 ppm for both of these sacrifice intervals while thyroid weight was increased at 75 ppm in males only after 52 weeks and in both sexes at 75 ppm after 104 weeks.

In conjunction with organ weight changes after 52 weeks of treatment, centrilobular hepatocyte hypertrophy was noted in females at 250 ppm, while follicular cell hypertrophy of the thyroid was noted in both sexes at 250 and 75 ppm. Slight to mild follicular cell hypertrophy was also observed at a low incidence (1 male and 2 females) at 20 ppm after 52 weeks. Both hepatocellular hypertrophy and follicular cell hypertrophy were completely reversible in females during the 13-week recovery period. Follicular cell hypertrophy was still present in the thyroid of males following recovery period, but compared to the 52-week sacrifice interval, a decrease in the percent incidence and severity of the lesion was noted indicating that reversibility was in progress. Other non-neoplastic histopathological changes in thyroid during the study involved the occurrence of thyroid gland colloid mineralisation in both sexes at 250 ppm from all sacrifice intervals (i.e. chronic, reversibility, and carcinogenicity phases) and in 75 ppm females from the carcinogenicity phase only as well as thyroid diffuse follicular cell hypertrophy in females at 250 ppm from the carcinogenicity phase. Mechanistic studies on the thyroid performed in a subsequent study clearly showed that dietary administration of the substance to rats resulted in elevated levels of circulating TSH due to an enhanced hepatic/biliary clearance of thyroxine (T4). Test substance-mediated induction of liver enzymes involved in T4 metabolism (e.g. γ-glucuronyl transferase) was associated with the increased clearance of T4, which would result in reduced circulating levels of T4 and thus a stimulation of TSH release from the pituitary. Since no thyroxine binding globulin (TBG) is present in the blood of rats, plasma T4 half-life is shorter in rats compared to humans, so that the increased turnover of thyroid hormone results in chronic stimulation of the thyroid by TSH. Therefore, the rat species is more sensitive to thyroid stimulation and the development of neoplastic changes than humans. Based on these data, the incidence of thyroid adenomas in the present study is not considered to be relevant for risk assessment in humans.

Histopathological lesions in liver at 250 ppm during the carcinogenicity phase included bile duct hyperplasia and sclerosis and focal sinusoidal dilatation in females, and eosinophilic and basophilic tigroid foci of cellular alteration in males. Furthermore, an increase in the severity, but not the incidence, of renal chronic nephropathy in both sexes at 250 ppm (from the carcinogenicity phase) associated with an increased incidence of arteritis/periarteritis in the kidney of females only at 250 ppm was observed. In females from the carcinogenicity phase, foci of alveolar macrophages in lungs were seen at 250 ppm. Chronic exposure during carcinogenicity phase (104 weeks), revealed proliferative (pre-neoplastic) changes consisting of slightly increased incidences (not statistically significant) of follicular cell focal hyperplasia and follicular cell adenomas of the thyroid in both sexes at 250 ppm as well as hepatocellular adenomas in males at 250 ppm. However, there was no indication of a statistically significantly, higher overall tumour incidence in either sex.

Overall, due to missing human relevance and/or limited toxicological significance as determined by missing statistical significance, the observed pre-neoplastic findings are not considered as conclusive for classification purposes. Based on the effects in thyroid and/or liver (histopathological changes, increased organ weights, and/or altered thyroid hormone or bilirubin levels), the NOAEL for chronic toxicity in rats was considered to be 0.85 mg/kg/day in males and 1.17 mg/kg/day in females (20 ppm in the diet, carcinogenic phase Weeks 1 -104). The NOAEL for pre-neoplastic lesions in thyroid and/or liver was 75 ppm in the diet, corresponding to 3.21 and 4.40 mg/kg bw/day in males and females, respectively.

 

In a further GLP-conform carcinogenicity study according to OECD guideline 451 and EPA OPPTS 870.4200, male and female C57BL/6 N Crl: BR mice were exposed to the test substance for a period of 78 weeks (M-205769-02-1, 2002). Fifty animals per sex and dose were administered the test substance at dietary levels of 10, 50, 150 and 300 ppm, corresponding to 1.7, 8.6, 25.6 and 50.8 mg/kg bw/day in males and 1.7, 12.5, 36.3 and 73.5 mg/kg bw/day in females, respectively. A further group of each 10 animals per sex for the control and high dose treatment was included for interim sacrifice at 52 weeks.

There were no treatment-related clinical signs noted at any dose level during either phase of the study. There was no evidence that treatment produced an increase in the incidence or reduction in the onset time of palpable tumours. The number and occurrence of unscheduled deaths was low and similar in all the control and treated groups, except for females of the 300 ppm group, where mortality was slightly higher (11/50) than in the control group (4/50). At study termination, a slight reduction of the survival rate for females given 300 ppm (-13% compared to controls) was observed compared to the control and the remaining dose groups. Although not statistically significant, this effect was considered to be related to treatment with the test substance.

The body weight gain was similar in the control and the treated groups for the males during the whole study period. A slight and transient reduction in body weight gain, which was not correlated to reduced food consumption, was observed in females treated with 50 ppm and above during the first 4 weeks of the study. Thereafter, body weight gain in these animals was similar to those of controls.

No toxicological relevant changes in haematological parameters were observed in animals of any treatment group compared to controls. Clinical chemistry at Week 52 revealed an increase in alanine aminotransferase activity (90 vs. 21 IU/L in controls) in males given 300 ppm. However, in the absence of macroscopic or microscopic changes in the liver, the relationship to treatment with the test substance was considered to be doubtful. Minor differences in absolute and relative organ weights were noted in the liver at 300 ppm after 52 or 78 weeks in both sexes. As the differences between treated and control males in the absolute and relative liver weights after 52 weeks were slight, and without correlative histopathological changes, they were considered to be of doubtful toxicological importance.

In females, a slight increase, but not dose-related increase in absolute and relative liver weights was also observed at 75 ppm. There were no treatment-related non-neoplastic changes in any organ at any of the dose-levels tested, but treatment with the test substance produced a slight increase (p = 0.0133) in the incidence of hepatocellular adenomas (HCA) in female mice at 300 ppm (6/50 vs. 0/50 in controls). However, these benign tumours were only observed in high dose females reaching the Maximum Tolerated Dose (MTD) where a reduced survival rate was observed. Since no treatment-related HCA were reported at the lower dose levels, this effect was thus devoid of any dose-relationship. In addition, no hepatocellular carcinoma was noted in either sex. Given the lack of genotoxicity potential, the absence of carcinoma following a prolonged exposure to the substance, and the absence of any dose-relationship, this increased incidence of HCA in high dose female mice was therefore considered to be subsequent to a threshold mechanism with a probable phenobarbital-like mechanism of action: hepatocellular hypertrophy associated with transient liver cell proliferation followed by a steady state. This assumption was further supported by subsequent, mechanistic studies in mice, showing that the test substance induced hepatic cell proliferation and cytochrome P-450 activities in liver with a phenobarbital-like profile.All other non-neoplastic and neoplastic lesions were recognised as those which are commonly occurring in mice of this strain and age and showed no indication of a treatment-relationship.

Based on the results of this study, the NOAEL for chronic toxicity in mice was set at 150 ppm (36.3 mg/kg bw/day) in females and ≥ 300 ppm (50.8 mg/kg bw/day) in males. For the occurrence of pre-neoplastic lesions in mice a LOAEL of 300 ppm (73.5 mg/kg bw/day) was established in females, whereas the NOAEL in males was ≥ 300 ppm (50.8 mg/kg bw/day).

 

 

Recently, thyroid perturbations and hepatocellular adenomas were assessed in detail to further elucidate the mode of action of those findings and to assess their human relevance.

Evidence from standard and explanatory studies suggests that the profile of observed liver effects are coherent with other molecules known to exert their effects through activation of the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) in the liver (M-767439-01-1). Other possible mode of actions for receptor and non-receptor mediated tumorigenesis for the liver have been dismissed because of a lack of plausibility and coherence. Hence, the 3 key events for CAR/PXR-mediated liver tumour mode of action have been considered: 1. CAR/PXR receptor activation in the liver: altered hepatic gene expression and/or induction of Phase I enzymes, 2. increased hepatocellular proliferation and 3. an increase in altered hepatic foci. Investigations in primary human and rat hepatocytes revealed that induction of key event 1 was much lower in human hepatocytes than that of reference compounds. In addition, no evidence of hepatocyte proliferation (key event 2) was observed in human hepatocytes when tested under the same in vitro conditions of test as those used for rat hepatocytes. The results indicate that the rodent liver tumours associated with ethiprole exposure is unlikely to occur in humans (for details, please refer to attachment M-767439-01-1 in section 13.2 of the technical dossier).

Also, tumor observations in the thyroid of rats are considered to be liver-mediated, initiated by activation of CAR/PXR nuclear receptors (M-767422-01-1). CAR/PXR activation results in an increase in the metabolic capacity of the liver and causes an increase in T4 clearance by induction of Phase II metabolic enzymes. Accordingly, a compensatory increase in serum TSH and TSH-dependent stimulation of the thyroid gland function and thyroid follicular cell proliferation is observed.

In order to address if the induction of thyroid tumors is also relevant for humans, 4 key events relevant for dose-response and temporal association have been assessed. Briefly, the following key events have been considered: 1. CAR/PXR receptor activation identified by induction of hepatic cytochrome P450 gene expression and enzyme activity, 2. Phase II liver enzyme induction leading to increased serum T4 clearance and consequently decreased circulating T4, 3. Increased TSH and 4. Increased thyroid cell proliferation.

 

Marked quantitative differences were observed between human and rat Phase I and Phase II enzyme activities, providing robust evidence that the mild rodent thyroid effects (hormone disruption and adenoma) are not relevant for humans (for details, please refer to attachment M-767422-01-1 in section 13.2 of the technical dossier).

 

Based on the available information it was concluded that the findings in liver and thyroid observed in rodent species are not relevant for humans. For details, please refer to section 13.2.

 

References:

M-767439-01-1: Ethiprole: Mode of Action Evaluation and Human Relevance Assessment of Rodent Hepatocellular Carcinomas (2021)

M-767422-01-1: Ethiprole: Mode of Action Evaluation and Human Relevance Assessment of Thyroid Pertubations in Rat (2021)