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

Endpoint summary

Description of key information

NOAEL (oral, rat, 28 d) = 9.2 (males) and 9.6 (females) mg/kg bw/day
NOAEL (oral, rat, 90 d) = 1.2 (males) and 1.5 (females) mg/kg bw/day

NOAEL (oral, rat, 52 weeks) = 0.98 (males) and 1.34 (females) mg/kg bw/day (chronic phase of the Combined Chronic Toxicity / Carcinogenicity study)

NOAEL (dermal, rat, 28 d) = 10 mg/kg bw/day

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Sep - 08 Oct 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

Current version adopted in 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

Guideline in place during study conduct: adopted in 1995

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.27 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Version / remarks:

Adopted in 1992

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Wistar (AF) RJ: WI (IOPS 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
- Body weight at study initiation: males: 158 - 203 g, females: 151 – 171 g
- Housing: individually, in suspended stainless steel wire mesh cages
- Diet: certified rodent powder diet "M 20 contrôlé" (Pietrement, Provins, France), ad libitum
- Water: filtered and softened water from the municipal water supply, ad libitum
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY: Routine analyses of feed and water indicated that there was no contamination which could have been expected to have compromised the study.

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: 07 Sep 1999 To: 08 Oct 1999

Route of administration:

oral: feed

Vehicle:

other: certified rodent powder diet

Remarks:

M 20 contrôlé

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): once for each concentration
- Mixing appropriate amounts with (Type of food): standard diet
- Storage temperature of food: -18 °C

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Method/Principle: Diet samples were extracted by maceration under pressure with hot toluene. The quantification was performed by Gas Chromatography (GC) using Electron Capture Detection (ECD) and external standardisation.

Homogeneity analysis: The lowest (20 ppm) and highest (2500 ppm) dose preparations were checked.

Concentration analysis: Mean values obtained from the homogeneity checks were used as measured concentrations. The concentration of the other preparations and a control diet sample were also checked.

Stability analysis: The stability of the test substance in the diet has been demonstrated in a previous study (90-day toxicity study in the rat by dietary administration, M-192116-02-2, 1997), where samples of 5 and 2500 ppm were analysed after preparation and after storage at room temperature for 7 weeks.

Results: All results for homogeneity and concentrations were within the target ranges of 85 to 115% of the nominal concentrations. The test substance was stable over a 52-day period at ambient temperature (approximately 20 °C) and under frozen condition (temperature below -15 °C).

Duration of treatment / exposure:

28 days

Frequency of treatment:

continuously, 7 days/week

Dose / conc.:

20 ppm

Remarks:

actual test substance intake: males: 1.8 mg/kg bw/day, females: 2.0 mg/kg bw/day

Dose / conc.:

100 ppm

Remarks:

actual test substance intake: males: 9.2 mg/kg bw/day, females: 9.6 mg/kg bw/day

Dose / conc.:

500 ppm

Remarks:

actual test substance intake: males: 46.1 mg/kg bw/day, females: 46.3 mg/kg bw/day

Dose / conc.:

2 500 ppm

Remarks:

actual test substance intake: males: 219.3 mg/kg bw/day, females: 220.2 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were selected after evaluation of the results of a sub-chronic toxicity study with the test substance (90-day toxicity study in the rat by dietary administration, M-192116-02-2, 1997).
- Fasting period before blood sampling for clinical biochemistry: Yes, animals were fasted overnight.

Observations and examinations performed and frequency:

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: detailed physical examinations were performed at least weekly during the treatment period. The nature, onset, severity, reversibility and duration of clinical signs were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed once during the acclimatisation period, on the first day of treatment, and then weekly throughout the treatment period and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during acclimatisation phase and during Week 3
- Dose groups that were examined: all animals (acclimatisation) and all surviving animals from the control and high dose groups (Week 3)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on study Days 23, 24 or 25, blood samples were taken by puncture of the retro-orbital venous plexus.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all surviving animals (98)
- Parameters listed in Table 1 under "Any other information on materials and methods incl. tables" were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On study Days 23, 24 or 25, blood samples were taken by puncture of the retro-orbital venous plexus.
- Animals fasted: Yes
- How many animals: all surviving animals (98)
- Parameters listed in Table 1 under "Any other information on materials and methods incl. tables" were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: On study days 29, 30, 31 or 32, in the morning, prior to sacrifice, overnight urine samples were collected.
- Animals fasted: Yes
- Parameters listed in Table 1 under "Any other information on materials and methods incl. tables" were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during the acclimatisation phase and during Week 3
- Dose groups that were examined: all
- Battery of functions tested: other: grasping, righting, corneal, pupillary, auditory startle, and head shaking reflexes

IMMUNOLOGY: No

OTHER: THYROID HORMONE ANALYSIS
- Time schedule for collection of blood: during Week 4 from the retro-orbital venous plexus of each surviving animal
- Animals fasted: Yes
- How many animals: all surviving animals (98)
- Parameters: thyrotropin = thyroid stimulating hormone (TSH), triiodothyronine (T3) and thyroxine (T4)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Table 2 under "Any other information on materials and methods incl. tables")
HISTOPATHOLOGY: Yes (see Table 2 under "Any other information on materials and methods incl. tables")

Necropsy was performed at Day 29, 30, 31, and 32 on all animals and included the examination of all major organs, tissues and body cavities. Macroscopic abnormalities were recorded, sampled and examined microscopically. Histopathological examinations were performed on all the animals, either found dead or killed by design, in the control and high dose groups as well as all decedents in the intermediate dose groups. Lung, liver, thyroid gland, kidney and adrenal gland were examined in the intermediate dose groups to identify the no effect level.

Statistics:

Mean values and standard deviations (SD) were calculated for each sex separately for each group at each time period. All statistical analysis were carried out separately for males and females.

Variables analysed:
- body weight parameters
- 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
- hormone data

Since the above parameters are all measured on a continuous scale, the treatment groups were compared using a combination of parametric ANOVA and nonparametric data analysis techniques. The homogeneity of variance assumption was examined via Bartlett's test. If Bartlett test rejects the homogeneity of variance assumption (p < 0.05), nonparametric tests were used. Nonparametric tests included the Kruskal-Wallis test for overall treatment group differences, followed by the Mann-Whitney U test for individual exposed vs. control group comparisons, if the Kruskal-Wallis test was significant (p < 0.05). All nonparametric tests were performed using the NPAR1WAY procedure in SAS® 6.12 (SAS Institute Inc., 1989, 1996).
If Bartlett's test does not reject the hypothesis of homogeneous variances, standard ANOVA techniques were applied for comparing the treatment groups. The GLM procedure in SAS® 6.12 (SAS Institute Inc., 1989, 1996) will be used to evaluate the overall effect of treatment, and, when a significant treatment effect is present (p < 0.05), to compare each exposed group to control via Dunnett's test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no treatment-related clinical signs of toxicity in animals surviving until study termination. Both males, which were found dead or sacrificed moribund, presented a general pallor before death.

Mortality:

mortality observed, treatment-related

Description (incidence):

3/100 mortalities occurred during the study. One male at 20 ppm died during anaesthesia for blood sampling. One male at 2500 ppm was found dead on Day 25 and one male at 2500 ppm was sacrificed moribund on Day 15.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight and body weight gain of male animals at 2500 ppm was statistically significantly decreased throughout the study period compared to control animal values (terminal body weight -8%). Females at 2500 ppm had a body weight gain lower during Week 1, but higher during Weeks 2 and 3 when compared to controls. At the end of the study, body weight in females treated at 2500 ppm was similar to controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 2500 ppm, food consumption was statistically significantly lower than controls during Weeks 1 and 2 in males and during Week 1 in females.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were noted at the ophthalmological examination.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematological examination showed statistically significantly increased mean prothrombin time in males at 500 ppm (+44%) and 2500 ppm (+120%). Statistically significantly higher mean platelet counts in both sexes were observed at 2500 ppm (+24% in males and +17% in females) (see Table 3 under "Any other information on results incl. tables"). These changes in coagulation parameters may have contributed to the macroscopic changes indicative of haemorrhage in deceased males of the highest dose group. In the absence of a dose-response relationship and since the individual values remained in the physiological range for rats of this strain and age, the decreased mean prothrombin time seen in females at 500 and 100 ppm was not considered to be biologically or toxicologically relevant.
Statistically significant changes were noted in erythrocyte parameters in both sexes. However, as the incidence of abnormal values and/or the amplitude relative to control were low, they were not considered toxicologically relevant. In the absence of relevant variation in total white blood cell counts, the statistically significant changes noted in differential counts (percentages and/or absolute counts) were not considered biologically or toxicologically relevant.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Higher mean total cholesterol concentrations were noted in both sexes at 2500 ppm and in females at 500 ppm (see Table 3 under "Any other information on results incl. tables"). In females, this change was associated with higher mean triglyceride concentrations. Higher mean alanine aminotransferase activity was observed in males at 2500 ppm. Mean total protein concentration was higher in females at 500 and 2500 ppm. In males treated at 2500 ppm, mean albumin concentration was lower. A slight non-toxicologically significant increase in total protein was observed in males at 2500 ppm and in females at 100 ppm. In the absence of a clear dose-response relationship, total bilirubin variations seen in both sexes were considered to be of no biological relevance. In view of the low degree and/or incidence, the other statistically significant changes noted (higher mean aspartate aminotransferase and alanine aminotransferase activities in males or females at 2500 ppm, lower mean glucose concentration in males at 2 500 ppm, lower mean chloride concentrations in females at 2500 and 500 ppm and higher mean calcium concentration in females at 2500 ppm) were judged to be of no toxicological relevance.

In summary, at 500 and 2500 ppm, increased cholesterol, triglycerides and protein values were observed in females. At 2500 ppm, increased alanine aminotransferase activity and cholesterol, and decreased albumin values were observed in males.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

In males, statistically significantly lower mean urinary volume was seen at 500 ppm (-41%) and 2500 ppm (-57%). In males of the high dose group, mean pH value was slightly reduced. Considering its low incidence, the lower mean urinary volume noted at 20 ppm in males was judged to be of no biological and toxicological relevance.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No effects were observed at the neurotoxicity assessment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Mean absolute and relative liver weights were statistically significantly and dose-dependently increased in males treated at 500 and 2500 ppm, and in females at 100, 500, and 2500 ppm when compared with the controls (see Table 4 under "Any other information on results incl. tables"). Mean absolute and relative thyroid gland weights were statistically significantly and dose-dependently higher in males and in females treated at 500 and 2500 ppm, when compared to controls. Mean absolute and relative adrenal gland weights were statistically significantly higher in males treated at 500 and 2500 ppm, and in females at 100 and 2500 ppm when compared with the controls. The change was slight and not statistically significant in females at 500 ppm. Statistically significantly lower absolute and relative prostate weights were found in males at 2500 ppm. This change was considered to have a doubtful significance, since no histological change was observed and in view of the lower terminal body weights. Lower absolute and relative spleen weights were observed in females at 500 and 2500 ppm, but they were not considered biologically significant according to the low level of change and the absence of any histological correlate. All other organ weight changes were incidental and were not related to treatment.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Enlargement (corresponding to the higher liver weights) and dark colour of the liver were found in almost all animals at 500 and 2500 ppm. A dark colour of the kidneys was observed in animals at 2500 ppm. Thyroid gland was enlarged in 2/8 males and 2/10 females treated at 2500 ppm. No other treatment-related macroscopic organ changes were seen at terminal sacrifice. All other macroscopic findings were judged to be incidental and not related to treatment. In animals with unscheduled deaths, dark appearance of the liver with multiple white foci as well as dark content in intestines was observed. One of these animals showed enlargement of the liver, which is consistent with the observations in animals at scheduled sacrifice. In the other dead animal, haemorrhage was noted around the genital area.

Neuropathological findings:

no effects observed

Description (incidence and severity):

Microscopical examination of the brain, one cranial nerve, and one peripheral nerve did not reveal any morphological change in the high dose group compared to the control group.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related organ changes were observed in the liver, the thyroid, the adrenal glands and the kidneys. A diffuse centrilobular to panlobular hepatocellular hypertrophy was observed in all males and in almost all females at 500 and 2500 ppm. Slight golden brown pigments were observed in the hepatocellular cytoplasm in 9/10 females at 2500 ppm. These changes were related to the enlargement and dark colour of the liver. Multifocal hepatocellular necrosis associated with mixed cell infiltrate was found in almost all groups at different incidence and at slight degree. This lesion was not considered to be treatment-related, since the same lesion was found in two controls at the same degree. In kidney, golden brown droplets were found in the cytoplasm of tubular epithelial cells in all males and in almost all females treated at 2500 ppm. They were related to the macroscopically dark colour of the kidney. A diffuse slight to moderate thyroid follicular cell hypertrophy was found in all males and all females treated at 500 and 2500 ppm. In the adrenal cortex, an increased diffuse vacuolation in the zona fasciculata and glomerulosa was observed in all males and females treated at 2500 ppm. A similar change was found in the zona fasciculata for 3/10 females and 3/10 males at 500 ppm and in the zona glomerulosa of 3/10 females at 500 ppm.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Thyroid hormone analysis showed significantly reduced levels of thyroxine (T4) associated with significantly higher levels of thyrotropin/ thyroid stimulating hormone (TSH) in males treated at 500 and 2500 ppm. In females, a slight and not statistically significant decrease was observed at 500 and 2500 ppm. Similar to males, levels of TSH were statistically significantly increased at 500 and 2500 ppm. No treatment-related effects on triiodothyronine (T3) were observed on Week 4 at any dose tested in both sexes, except for a slight, but physiologically insignificant increase in T3 levels at 2500 ppm.

Key result

Dose descriptor:

NOAEL

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: actual test substance intake: males: 9.2 mg/kg bw/day, females: 9.6 mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

urinalysis

other: see 'Remark'

Remarks on result:

other: actual test substance intake: males: 46.1 mg/kg bw/day, females: 46.3 mg/kg bw/day

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 ppm

System:

other: hepatobiliary, urinary, and endocrine system

Organ:

adrenal glands

kidney

liver

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Table 3. Changes in clinical chemistry parameters (data are presented as mean values ± SEM)

Parameters	Test substance [ppm]
	0	20	100	500	2500
PLT [10E+09/L)
male	1245 ± 44	1234 ± 34	1156 ± 107	1359 ± 47	1542 ± 81* (a)
female	1260 ± 67	1269 ± 44	1353 ± 35	1356 ± 85	1475 ± 37** (a)
PT [s]
male	12.4 ± 0.1	12.8 ± 0.2	12.9 ± 0.2	17.8 ± 0.4*** (a)	27.3 ± 1.8*** (a)
female	13.5 ± 0.3	12.7 ± 0.2	12.0 ± 0.3** (a)	12.00 ± 0.2** (a)	14.7 ± 0.8
CHOL [mmol/L]
male	1.90 ± 0.08	1.76 ± 0.09	1.82 ± 0.11	2.05 ± 0.07	4.03 ± 0.40*** (a)
female	2.15 ± 0.08	2.22 ± 0.12	2.30 ± 0.12	3.64 ± 0.26*** (a)	5.31 ± 0.27*** (b)
TRIG [mmol/L]
male	0.99 ± 0.14	0.89 ± 0.10	0.84 ± 0.11	0.91 ± 0.09	1.27 ± 0.19
female	0.43 ± 0.05	0.44 ± 0.03	0.38 ± 0.03	0.79 ± 0.09** (a)	1.49 ± 0.39** (a)
ALAT [IU/L]
male	23.1 ± 1.0	21.6 ± 1.5	23.3 ± 1.3	27.3 ± 2.1	107.1 ± 39.4*** (a)
female	20.6 ± 1.2	20.4 ± 0.8	21.3 ± 1.3	31.4 ± 2.7** (a)	32.7 ± 1.6*** (a)
TPRO [g/L]
male	63.5 ± 1.0	61.6 ± 0.6	62.8 ± 1.0	65.6 ± 1.1	68.1 ± 1.1** (b)
female	58.4 ± 0.5	60.4 ± 1.1	62.5 ± 0.8* (b)	67.9 ± 1.3*** (b)	66.1 ± 1.3*** (b)
ALB [g/L]
male	33.9 ± 0.5	33.8 ± 0.2	33.4 ± 0.4	33.1 ± 0.4	30.9 ± 0.5*** (b)
female	33.0 ± 0.3	33.6 ± 0.6	34.3 ± 0.5	33.8 ± 0.6	32.0 ± 0.5
TBIL [µmol/L]
male	1.55 ± 0.14	1.61 ± 0.16	1.01 ± 0.13* (b)	1.01 ± 0.12* (b)	1.32 ± 0.08
female	1.58 ± 0.14	1.59 ± 0.14	1.09 ± 0.12* (b)	0.93 ± 0.10** (b)	1.00 ± 0.07** (b)
GLUC [mmol/L]
male	7.45 ± 0.45	7.77 ± 0.33	7.85 ± 0.55	6.39 ± 0.50	5.57 ± 0.28* (b)
female	5.99 ± 0.31	5.54 ± 0.20	5.67 ± 0.33	6.22 ± 0.27	5.45 ± 0.27
CL [mmol/L]
male	103.3 ± 0.6	104.0 ± 0.5	103.3 ± 0.4	102.8 ± 0.5	102.6 ± 0.3
female	106.1 ± 0.4	104.9 ± 0.6	105.7 ± 0.6	103.7 ± 0.5* (b)	102.8 ± 0.6*** (b)
CA [mmol/L]
male	2.73 ± 0.03	2.71 ± 0.01	2.72 ± 0.03	2.78 ± 0.03	2.80 ± 0.02
female	2.60 ± 0.03	2.63 ± 0.03	2.62 ± 0.03	2.70 ± 0.03	2.76 ± 0.04** (b)

PT = prothrombin time; PLT = platelet count; CHOL = cholesterin; TRIG = triglycerides; ALAT = alanine transferase; TPRO = total protein: ALB = albumin; TBIL = total bilirubin; GLUC = glucose; CL = chloride; CA = calcium; SEM = standard error of the mean

(a) Kruskal-Wallis test followed by Mann-Whitney U Test for pairwise comparisons to control

(b) ANOVA test followed by Dunnett's test for pairwise comparisons to control

Statistical significance is indicated by *p < 0.05, **p < 0.01 and ***p < 0.001.

Table 4. Treatment-related changes in organ weights (data are presented as mean values ± SEM)

	LIVER
	absolute organ weight [g]		relative organ weight [% of terminal body weight]
Dose (ppm)	males	females	males	females
0	10.27 ± 0.42	5.98 ± 0.14	3.03 ± 0.08	2.85 ± 0.05
20	10.06 ± 0.21	6.27 ± 0.12	2.96 ± 0.05	2.96 ± 0.06
100	10.31 ± 0.15	7.20 ± 0.13*** (a)	3.10 ± 0.07	3.35 ± 0.04*** (a)
500	13.90 ± 0.25*** (a)	11.74 ± 0.43*** (a)	4.16 ± 0.06*** (b)	5.31 ± 0.16*** (a)
2500	20.38 ± 0.32*** (a)	15.64 ± 0.59*** (a)	6.58 ± 0.12*** (b)	7.43 ± 0.21*** (a)
	THYROID GLAND
	absolute organ weight [g]		relative organ weight [% of terminal body weight]
	males	females	males	females
0	0.017 ± 0.001	0.015 ± 0.001	0.0049 ± 0.0002	0.0072 ± 0.0006
20	0.018 ± 0.002	0.016 ± 0.001	0.0053 ± 0.0005	0.0076 ± 0.0004
100	0.019 ± 0.002	0.014 ± 0.001	0.0058 ± 0.0005	0.0067 ± 0.0006
500	0.024 ± 0.002*** (a)	0.021 ± 0.001** (b)	0.0073 ± 0.0005*** (a)	0.0095 ± 0.0005* (b)
2500	0.026 ± 0.003** (a)	0.024 ± 0.001*** (b)	0.0084 ± 0.0007** (a)	0.0113 ± 0.0006*** (b)
	ADRENAL GLAND
	absolute organ weight [g]		relative organ weight [% of terminal body weight]
	males	females	males	females
0	0.057 ± 0.003	0.060 ± 0.003	0.017 ± 0.001	0.029 ± 0.001
20	0.052 ± 0.002	0.066 ± 0.002	0.015 ± 0.001	0.031 ± 0.001
100	0.054 ± 0.001	0.074 ± 0.004** (b)	0.016 ± 0.000	0.035 ± 0.002* (b)
500	0.066 ± 0.002** (b)	0.069 ± 0.003	0.020 ± 0.001** (b)	0.031 ± 0.001
2500	0.070 ± 0.003*** (b)	0.081 ± 0.003*** (b)	0.022 ± 0.001*** (b)	0.039 ± 0.001*** (b)

SEM = standard error of the mean

(a) Kruskal-Wallis test followed by Mann-Whitney U Test for pairwise comparisons to control

(b) ANOVA test followed by Dunnett's test for pairwise comparisons to control

Statistical significance is indicated by *p < 0.05, **p < 0.01 and ***p < 0.001.

Conclusions:

Treatment of rats for 28 days at dose levels of 20, 100, 500, and 2500 ppm with the test substance, resulted in toxicological effects on body weight, haematology, clinical chemistry, hormone levels, organ weights and tissue morphology at 500 and 2500 ppm. The liver, thyroid, adrenal gland, and kidneys were affected. Based on the results, the NOAEL was 100 ppm, equating to an actual test substance intake of 9.2 and 9.6 mg/kg bw/day in males and females, respectively.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Sep - 12 Dec 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

Current version adopted in 2018

Deviations:

yes

Remarks:

Oestrus cycle was not determined.

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

Guideline in place during study conduct: adopted in 1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

Adopted in 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

Version / remarks:

Adopted in 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (M.A.F.F.), notification 59 NohSan N° 4200

Version / remarks:

Adopted in 1985

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

(AF) RJ: WI (IOPS 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 - 7 weeks
- Body weight at study initiation: males: 243 - 280 g, females: 174 - 203 g
- Housing: individually, in suspended stainless steel wire mesh cages
- Diet: certified rodent powder diet "M 20 contrôlé" (Pietrement, Provins, France), ad libitum
- Water: filtered and softened water from the municipal water supply (drinking water quality), ad libitum
- Acclimation period: 14 days

DETAILS OF FOOD AND WATER QUALITY
Routine analyses of food and water indicated that there was no contamination, which could have compromised the study.

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: 10 Sep 1997 To: 09 - 12 Dec 1997

Route of administration:

oral: feed

Vehicle:

other: rodent powder diet

Remarks:

M 20 contrôlé

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): every 3 weeks for each concentration
- Mixing appropriate amounts with (Type of food): standard rodent powder diet
- Storage temperature of food: -18 °C

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Method/principle: The test substance was extracted from diet by homogenization with acetonitrile. The quantification was performed by High Performance Liquid Chromatography (HPLC) on a C18 column using ultraviolet (UV) detection at 277 nm and external standardization.

ANALYTICAL PRE-STUDY
Diet formulations were prepared once during the course of the pre-study at 2 concentrations (5 and 2500 ppm). Mixtures were stored frozen at a temperature below -15 °C and allowed to reach room temperature before sampling and use. Each sample weighed approximately 30 g of which 10 g were analysed according to the above analytical method.
- Homogeneity analysis: The lowest (5 ppm) and highest (2500 ppm) dose preparations were checked.
- Concentration analysis: Mean values obtained from the homogeneity checks were used as measured concentrations.
- Stability analysis: The 5 ppm and 2500 ppm diet formulation stability was checked once after 10, 17, 24, 31 and 45 days at a temperature below -15 °C, followed by a 1-week period at ambient temperature (approximately 20 °C) and once after 17, 24, 31, 38 and 52 days at ambient temperature (approximately 20 °C). At each sampling day, control samples were also taken.
- Results: Homogeneity and concentration results were within target ranges (85 - 115% of nominal concentration). Results of all control samples were below the quantification limit. Stability results were within target ranges, except those of the 5 ppm preparation after 52-day storage that were slightly below ranges (84% instead of 85%). According to the available results on homogeneity, concentrations and stability of the test substance in ground rodent, diet mixtures were declared acceptable during the pre-study.

ANALYTICAL STUDY
Diet formulations were prepared 4 times during the course of the animal study at 4 concentrations (5, 20, 500 and 2500 ppm). Homogeneity and concentrations were checked according to the above analytical method. Mixtures were stored frozen at a temperature below -15 °C and allowed to reach room temperature before sampling and use. Each sample weighed approximately 30 g of which 10 g were analysed.
- Homogeneity analysis: The 5 ppm and 2500 ppm (first formulation sequence) were checked.
- Concentration analysis: The concentration of the preparations from the first, second and fourth formulations was checked. At each sampling day, control samples were also taken.
- Results: All the homogeneity and concentration results were within target ranges (85 - 115% of nominal concentration). Results of all control samples were below the quantification limit. According to the available results, homogeneity and concentrations of the test substance in ground rodent diet mixtures were acceptable during this study.

Duration of treatment / exposure:

90 days

Frequency of treatment:

continously, 7 days/week

Dose / conc.:

5 ppm

Remarks:

actual test substance intake: males: 0.296 mg/kg bw/day, females: 0.373 mg/kg bw/day

Dose / conc.:

20 ppm

Remarks:

actual test substance intake: males: 1.174 mg/kg bw/day, females: 1.503 mg/kg bw/day

Dose / conc.:

500 ppm

Remarks:

actual test substance intake: males: 30.484 mg/kg bw/day, females: 37.567 mg/kg bw/day

Dose / conc.:

2 500 ppm

Remarks:

actual test substance intake: males: 154.746 mg/kg bw/day, females: 187.867 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were selected after evaluation of the results of a previous 90-day dietary study in Sprague Dawley rats performed with this test substance (M-210434-01-1, 1996).
- Fasting period before blood sampling for clinical biochemistry: Animals were fasted overnight prior to blood sampling.

Observations and examinations performed and frequency:

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations were performed at least weekly during the treatment period. The nature, onset, severity, reversibility and duration of clinical signs were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed once during the acclimatisation period, on the first day of treatment, and then weekly throughout the treatment period and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during acclimatisation phase and during Week 12
- Dose groups that were examined: all animals (acclimatisation) and all surviving animals from the control and high dose groups (Week 12)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On Study Day 85, 86 or 87, blood samples were taken by puncture of the retro-orbital venous plexus.
- Anaesthetic used for blood collection: Yes, isoflurane.
- Animals fasted: Yes
- How many animals: all surviving animals (87/100)
- Parameters listed in Table 1 under "Any other information on materials and methods incl. tables" were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On Study Day 85, 86 or 87, blood samples were taken by puncture of the retro-orbital venous plexus.
- Animals fasted: Yes
- How many animals: all surviving animals (87/100)
- Parameters listed in Table 1 under "Any other information on materials and methods incl. tables" were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: On Study Day 91, 92, 93 or 94, in the morning, prior to sacrifice, overnight urine samples were collected.
- Animals fasted: Yes
- Parameters listed in Table 1 under "Any other information on materials and methods incl. tables" were examined.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER: THYROID HORMONE ANALYSIS
- Time schedule for collection of blood: at Week 2, 6 and 13 from the retro-orbital venous plexus of each surviving animal
- Animals fasted: Yes
- How many animals: all surviving animals (87/100)
- Parameters: thyrotropin = thyroid stimulating hormone (TSH), triiodothyronine (T3), and thyroxine (T4)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Table 2 under "Any other information on materials and methods incl. tables")
HISTOPATHOLOGY: Yes (see Table 2 under "Any other information on materials and methods incl. tables")

Necropsy was performed at Days 91, 92, 93 and 94 on all animals and included the examination of all major organs, tissues and body cavities. Macroscopic abnormalities were recorded, sampled and examined microscopically. Histopathological examinations were performed on all the animals, either found dead or killed by design, in the control and the 500 and 2500 ppm dose groups, all decedents in all dose groups, and on lung, liver, testis, epididymis, thyroid gland, kidney and adrenal gland of all animals in the study. Target organs were examined in the intermediate dose groups as necessary to identify the no effect level.

Statistics:

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

Variables analysed:
- body weight parameters
- 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
- hormone data

Results of the variables analysed were intercompared for the exposed groups and the control group by use of Bartlett’s test for homogeneity of variances and analysis of variance (ANOVA). If Bartlett’s test indicated homogeneous variances and the ANOVA was significant, the group mean values were intercompared to the control mean value using the Dunnett’s test. If Bartlett’s test indicated heterogeneous variances, non-parametric statistical procedure was performed using the Kruskal-Wallis non-parametric one-way analysis of variance by ranks. If the Kruskal-Wallis test was significant, the Mann-Whitney test was used to compare each group to the control. The levels of significance for each statistical comparison were 0.05 and 0.01.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 2500 ppm, piloerection, increased and/or reduced motor activity and irritability to touch were observed. These signs were observed on only very few occasions during the study. Irritability to touch was also observed occasionally in all other groups including the controls with no dose-response relationship. In addition, most of the males, which died at 2500 ppm, exhibited a general pallor before death.

Mortality:

mortality observed, treatment-related

Description (incidence):

In total, 13/100 animals died during the study.
At 2500 ppm, 7/10 male animals were found dead and one male was sacrificed in moribund condition.
At 500 ppm, one male and one female were sacrificed in moribund condition, one female was found dead (both treatment-related). One female died during anaesthesia for blood sample on Week 6 (regarded as not treatment-related).
At 5 ppm, one female was sacrificed in moribund condition.
All these mortalities occurred between Days 19 and 90. The mortalities observed in animals treated at 500 or 2500 ppm were considered related to treatment. The single mortality observed in females at 5 ppm was considered incidental, since no mortality in either sex was observed at the next higher dose level. In addition, data from a subsequent 52-week rat study with 70 animals/sex/group showed no increased incidence of mortality in males and no mortality at all in females, at 5 or 20 ppm, during the 52-week treatment period with the test substance.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain of males at 2500 ppm was significantly decreased compared to controls during the first 5 weeks of the study. This difference tended to decrease thereafter. In females, the body weight gain at 2500 ppm was significantly decreased compared to controls during the first week only. There were no changes in terminal body weights of treated males or females when compared with controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 2500 ppm, a statistically significant lower food consumption was noted in males during the first 3 weeks and in females during the first week of the study.

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 observed at the ophthalmological examination.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At 2500 and 500 ppm, statistically significant decreases were observed in red blood cell parameters in both sexes (see Table 3 under "Any other information on results incl. tables").
Treatment-related differences were seen in prothrombin time at 2500 or 500 ppm in males only, mean values were higher by 165% (not statistically analysed) and 44% (α = 0.01), respectively when compared to control values.
A tendency towards higher platelet counts was noted at 2500 ppm; the mean value was 28% higher in males (not statistically analysed), and 22% higher in females (α = 0.05) when compared to control values. The other statistically significant changes observed were judged to be not biologically relevant, as no clear dose-response relationship was evident. In addition, for leukocyte parameters, no statistically significant variation was noted in total white blood cell counts.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

A treatment-related increase in total cholesterol and total protein concentrations was observed (see Table 3 under "Any other information on results incl. tables"). Cholesterol was increased in males at 2 500 ppm and in females at 500 and 2 500 ppm. Protein concentrations were increased in males and females at 500 and 2 500 ppm. In females, higher mean total cholesterol concentrations were associated with higher mean triglycerides concentrations (+41%, α = 0.05 and +37%, not statistically significant, respectively) at 2500 and 500 ppm. In males at 2500 ppm, the higher mean total protein concentration was associated with an increased mean albumin concentration (+9%, not statistically analysed). In males at 2500 ppm, increased mean alanine aminotransferase activity was observed (+156%, not statistically analysed) compared to controls. Calcium concentrations were increased in males and females at 500 and 2 500 ppm. Potassium was increased in both sexes at 2 500 ppm. Chloride was decreased in females at 500 and 2 500 ppm. The other statistically significant differences seen in some treated groups, when compared with controls, were not considered to be biologically or toxicologically relevant, as no dose-response relationship was seen. No meaningful variations were noted in the other parameters analysed.
In summary, at 500 and 2500 ppm, increased total protein (male, female), calcium (male, female) and cholesterol (female) and decreased chloride (female) values were observed. At 2500 ppm, increased potassium (male, female) and cholesterol (male) values were reported.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No treatment related effects were noted in the parameters analysed in this 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):

Statistically significant organ weight increases were noted in the liver and thyroid in both sexes at 500 and 2500 ppm. In both males and females, a dose-related increase in liver weights (absolute and relative) was observed from 500 ppm when compared to control values. No effect on liver weight was observed in the 20 or 5 ppm dose groups. In males and females, a dose-related increase in both absolute and relative thyroid weights was observed from 500 ppm when compared to control values. No effect on thyroid weight was observed in the 20 or 5 ppm dose groups. Statistically significant higher mean absolute and/or relative heart weights were seen in males and females at 2500 ppm and in males at 500 ppm, but were not considered to be treatment-related in the absence of corroborative histological changes in this organ. In females at 2500 ppm, there were statistically significantly higher mean absolute and relative adrenal weights. At 500 ppm the mean adrenal to body weight ratio was also statistically significantly higher, when compared with controls. However, this was considered unlikely to be related to treatment since the difference was slight and there were no treatment-related histological changes. All other organ weight changes in males and females were thought to be incidental and not related to treatment.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Unscheduled deaths:
8/10 males at 2500 ppm, 1/10 males and 3/10 females at 500 ppm and a 1/10 females at 5 ppm died or were sacrificed in a moribund condition during the study. Evidence of recent haemorrhage in one or more sites in the body was observed in these animals. There was a tendency towards increased pallor in males at 2500 ppm. Significant macroscopic changes were also seen in the liver of all the males and the majority of the decedent females. All males at 2500 ppm and the single male at 500 ppm had liver enlargement with or without rounded edges. Liver colour was also abnormal having a green tinge (sometimes associated with dark liver colour), or a pale, abnormal colour in the majority of rats. Red and/or white foci were also seen in the majority of these livers. Two females at 500 ppm (including one, which died during anaesthesia) also had an enlarged liver. Dark kidney colour was seen in the majority of males at 2500 ppm and in a single male and female at 500 ppm. Thyroid enlargement was noted in two decedent males at 2500 ppm and in a single decedent female at 500 ppm.

- Terminal sacrifice:
Significant macroscopic changes were seen in the liver and kidney of the majority of males and females at 2500 and 500 ppm. Liver enlargement and dark liver colour (often with a green tinge), were seen in all surviving males at 2500 ppm and the majority of females at 2500 ppm as well as in both sexes at 500 ppm. Dark kidney colour was seen in all males and females at 2500 ppm and a proportion of males and females at 500 ppm. Thyroid enlargement was noted in single males at 2500 and 500 ppm and in a proportion of females at these treatment levels, at final sacrifice, when compared with controls.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At the histopathological examination, the liver with centrilobular to generalised hepatocyte hypertrophy and the thyroid with follicular hypertrophy/hyperplasia were found to be the target organs in animals at 500 and 2500 ppm. (Please see Tables 5 and 6 under "Any other information on results incl. tables" for details.)

- Unscheduled deaths:
There were thirteen unscheduled deaths during the study, the majority of which were males at 2500 ppm. In the majority of unscheduled deaths, there was evidence of recent and/or older haemorrhage in one or more sites. In one male found dead at 2500 ppm no evidence of haemorrhage was found, but severe extramedullary haemopoiesis in the spleen and prominent erythropoiesis in the bone marrow was observed. Centrilobular hepatocyte necrosis was also observed in these animals treated at 2500 ppm. Generalised hepatocyte hypertrophy was seen in the majority of males at 2500 ppm and in a single male and female at 500 ppm, when compared with controls. Golden brown cytoplasmic pigment was seen in three males out of eight examined at 2500 ppm and in a single female out of three examined at 500 ppm. Thyroid follicular epithelial hypertrophy/hyperplasia was seen in the majority of decedent males at 2500 ppm and decedent females at 500 ppm, and in the single decedent male at 500 ppm, when compared with controls. Mild to moderate amounts of golden brown pigment was seen within renal cortical tubules in a proportion of decedent males at 2500 ppm and the single decedent male at 500 ppm. The majority of decedent males at 2500 ppm had a higher degree of splenic extramedullary haemopoiesis, when compared with controls. Adrenal cortical hypertrophy/hyperplasia was seen in the majority of decedent males and in a single decedent female at 500 ppm, when compared with controls. Mild to marked lymphoid atrophy/apoptosis was seen in the thymus of the majority of decedent males at 2500 ppm. Reduced or absent colloid in the male secondary sex organs was seen in the majority of decedent males at 2500 ppm, when compared with controls. The adrenal, thymic and secondary sex organ changes were considered to be a result of stress and/or the debilitated condition of these animals and not directly related to treatment. In addition, mild to marked lymphoid hyperplasia was seen in the mesenteric lymph nodes of the majority of decedent males at 2500 ppm, when compared with controls but in the absence of other related findings, the significance of this finding is uncertain.

- Terminal sacrifice
Significant changes were noted in the liver and thyroid.
Treatment-related centrilobular to generalised hepatocellular hypertrophy, which showed an apparent dose-response relationship, was seen in the liver of both sexes at 2500 and 500 ppm, when compared with controls. These changes correlated with the increase in liver weights and were generally more pronounced in females. Mild to moderate amounts of golden brown cytoplasmic pigment were seen within the sinusoidal lining cells and occasionally also within hepatocyte cytoplasm in the majority of females at 2500 ppm. Mild amounts of this pigment were also seen in the two surviving males at 2500 ppm and occasional females at 500 ppm. Single cell necrosis was seen in one of the two surviving males at 2500 ppm and in two females at 500 ppm. Similar changes were not seen in either sex at 20 or 5 ppm. Mild to marked thyroid follicular epithelial hypertrophy/hyperplasia was seen in all animals at 2500 ppm, and in all males and the majority of females at 500 ppm at terminal sacrifice, when compared with controls. In females, there was an apparent dose-related increase in degree. These changes correlated with the increase in thyroid gland weights. Slight follicular epithelial hypertrophy/hyperplasia was seen in a single male at 20 and 5 ppm, and mild follicular epithelial hypertrophy/hyperplasia in two males at 20 ppm, but these findings were not considered to be toxicologically significant in view of the low level of change and the small number of animals involved and the absence of corroborative changes in thyroid weights or thyroid hormone levels. An increased degree of cortical tubular golden brown pigment was seen in the kidneys of all surviving males and females at 2500 ppm, when compared with controls. No histological changes were seen in the heart, which would account for the higher recorded organ weights in both sexes at 2500 ppm and males at 500 ppm. This was considered likely to be associated with dilated heart ventricles containing blood rather than a treatment-related effect. All other changes were considered to be incidental and not related to treatment.

In conclusion, the liver with centrilobular to generalized hepatocyte hypertrophy and the thyroid with follicular hypertrophy/hyperplasia were found to be the target organs in animals at 500 and 2500 ppm. The kidney was also affected at 2500 ppm with both sexes showing tubular golden brown pigment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Thyroid hormone analysis in males showed a significant decrease in T4 hormone levels associated with a marked increase in the levels of TSH at Weeks 2, 6 and 13 in the 500 and 2500 ppm groups (see Table 4 under "Any other information on results incl. tables"). In females treated at 500 or 2500 ppm, hormonal changes consisted of a decrease in T4 levels, but only at Week 6 associated with an increase in TSH levels. An increase in TSH plasma levels was also observed at Week 2 and 13, but without correlation to the decrease in T4 levels. In addition, a slight increase in T3 levels was observed at 2500 ppm in females at Weeks 6 and 13 and in males at Week 13. No treatment-related thyroid hormone changes were observed at 5 or 20 ppm.

Key result

Dose descriptor:

NOAEL

Effect level:

20 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no mortality, no changes in thyroid hormone levels and clinical chemistry parameters, no effects on liver and thyroid weights and morphology

Remarks on result:

other: actual test substance intake: males: 1.174 mg/kg bw/day, females: 1.503 mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

other: see 'Remark'

Remarks on result:

other: actual test substance intake: males: 30.484 mg/kg bw/day, females: 37.567 mg/kg bw/day

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 ppm

System:

other: hepatobiliary and endocrine system

Organ:

liver

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Table 3. Changes in haematology and clinical chemistry parameters (data are presented as mean values ± SD)

Parameters	Test substance [ppm]
	0	5	20	500	2500
Haematology	n = 9/9/10	n = 10/8/10	n = 10/9/10	n = 9/7/10	n = 4/10/10
HGB [g/100 mL]
male	15.8 ± 0.5	16.0 ± 0.5	15.6 ± 0.8	15.4 ± 0.6	14.6 ± 0.4
female	15.4 ± 0.7	15.3 ± 0.5	15.3 ± 0.4	14.4 ± 0.6**	13.8 ± 0.4**
HCT [%]
male	0.47 ± 0.02	0.49 ± 0.01	0.47 ± 0.02	0.46 ± 0.02	0.46 ± 0.02
female	0.46 ± 0.02	0.45 ± 0.02	0.45 ± 0.01	0.43 ± 0.02*	0.42 ± 0.01**
MCV [fL]
male	50.6 ± 1.1	50.8 ± 1.4	51.3 ± 1.8	48.1 ± 0.9**	47.8 ± 1.7
female	52.3 ± 1.3	51.1 ± 1.1	52.0 ± 1.3	48.4 ± 1.3**	47.3 ± 1.0**
MCH [pg]
male	16.9 ± 0.5	16.7 ± 0.4	17.1 ± 0.6	15.9 ± 0.3**	15.3 ± 0.4
female	17.7 ± 0.5	17.4 ± 0.3	17.6 ± 0.5	16.1 ± 0.6**	15.5 ± 0.3**
MCHC [g/100 mL]
male	33.5 ± 0.6	32.9 ± 0.5*	33.2 ± 0.4	33.3 ± 0.4	32.0 ± 0.3
female	33.9 ± 0.4	34.2 ± 0.4	33.9 ± 0.5	33.4 ± 0.4	32.8 ± 0.4**
PLT [10E+09/L)
male	1037 ± 153	1039 ± 127	1016 ± 162	1162 ± 98	1329 ± 194
female	1059 ± 112	1051 ± 182	1085 ± 154	1228 ± 237	1289 ± 180*
PT [s]
male	15.8 ± 0.9	15.2 ± 1.1	15.2 ± 0.9	22.7 ± 3.1**	41.8 ± 10.7
female	16.4 ± 1.2	15.6 ± 1.3	15.6 ± 0.7	14.2 ± 1.5	17.5 ± 5.4
Clinical chemistry	n = 10/10/10	n = 10/9/10	n = 10/10/10	n = 9/7/10	n = 4/10/10
CHOL [mmol/L]
male	2.09 ± 0.41	2.28 ± 0.50	2.39 ± 0.41	2.14 ± 0.59	3.01 ± 0.51
female	2.15 ± 0.34	2.13 ± 0.34	2.46 ± 0.33	4.60 ± 1.07**	7.16 ± 1.62**
TRIG [mmol/L]
male	0.90 ± 0.33	0.81 ± 0.16	0.76 ± 0.16	0.97 ± 0.43	1.00 ± 0.08
female	0.46 ± 0.11	0.48 ± 0.10	0.51 ± 0.12	0.63 ± 0.14	0.65 ± 0.22*
ALAT [IU/L]
male	31.5 ± 5.0	58.3 ± 84.8	31.5 ± 5.3	32.6 ± 4.8	80.5 ± 35.3
female	29.1 ± 5.0	26.3 ± 4.9	39.8 ± 32.1	36.9 ± 13.5	41.0 ± 13.7
AP [IU/L]
male	68.6 ± 8.3	68.0 ± 14.2	64.9 ± 14.5	62.8 ± 8.6	59.5 ± 12.71
female	43.4 ± 7.6	44.4 ± 12.4	47.0 ± 19.4	25.0 ± 5.5*	28.6 ± 4.4**
TPRO [g/L]
male	69.6 ± 3.1	69.2 ± 2.6	68.4 ± 3.0	75.0 ± 4.9**	84.0 ± 4.2
female	66.5 ± 3.9	67.2 ± 2.6	67.4 ± 2.8	78.9 ± 3.7**	83.2 ± 3.6**
ALB [g/L]
male	33.4 ± 0.7	33.4 ± 1.1	33.0 ± 1.3	34.7 ± 2.1	36.5 ± 2.1
female	34.5 ± 2.5	34.7 ± 2.1	33.7 ± 2.3	36.7 ± 1.5	35.2 ± 1.6
CL [mmol/L]
male	104.8 ± 1.5	104.8 ± 1.5	105.3 ± 1.0	103.7 ± 1.5	103.5 ± 0.6
female	107.1 ± 1.2	105.6 ± 1.7	105.9 ± 1.7	103.6 ± 1.3**	102.6 ± 1.6*
CA [mmol/L]
male	2.70 ± 0.09	2.69 ± 0.06	2.69 ± 0.08	2.80 ± 0.13*	2.99 ± 0.09
female	2.62 ± 0.12	2.63 ± 0.07	2.63 ± 0.08	2.78 ± 0.09**	2.89 ± 0.09**
K [mmol/L]
male	3.90 ± 0.31	3.78 ± 0.34	3.80 ± 0.25	3.99 ± 0.12	4.25 ± 0.21
female	3.50 ± 0.29	3.38 ± 0.44	3.40 ± 0.13	3.40 ± 0.22	3.79 ± 0.24*

HBG = haemoglobin; HCT = haematocrit; MCV = mean corpuscular volume; MCH = mean corpuscular haemoglobin; MCHC = Mean corpuscular haemoglobin concentration; PLT = platelet count; PT = prothrombin time; CHOL = cholesterin; TRIG = triglycerides; ALAT = alanine transferase; AP = alkaline phosphatase; TPRO = total protein: ALB = albumin; CL = chloride; CA = calcium; K = potassium; SD = standard deviation

n = x/y/z = number of males/females/total animals per group

The mean value of the test group was significantly different from control with Dunnett's or Mann-Whitney's test of significance at significance levels of * α = 0.05 and ** α = 0.01. Due to the small sample number (n = 4), mean values of males from the high dose group (2500 ppm) were not statistically analysed.

Table 4. Treatment-related changes in thyroid hormones (data are presented as mean values ± SD)

Parameters	Test substance [ppm]
	0	5	20	500	2500
Week 2	n = 10/10/10	n = 10/10/10	n = 10/10/10	n = 10/10/10	n = 10/10/10
TSH [ng/L]
male	0.57 ± 0.08	0.65 ± 0.18	0.62 ± 0.14	1.13 ± 0.36**	1.17 ± 0.27**
female	0.48 ± 0.10	0.42 ± 0.05	0.48 ± 0.10	0.76 ± 0.19**	0.97 ± 0.68**
T3 [nmol/L]
male	0.78 ± 0.21	0.72 ± 0.23	0.80 ± 0.20	0.77 ± 0.19	0.89 ± 0.23
female	0.81 ± 0.20	0.81 ± 0.21	0.89 ± 0.27	0.81 ± 0.15	0.79 ± 0.34
T4 [nmol/L]
male	36.2 ± 5.3	36.5 ± 4.6	37.7 ± 5.2	28.6 ± 2.9**	20.3 ± 2.4**
female	28.3 ± 5.3	29.1 ± 8.7	28.1 ± 6.2	25.5 ± 2.9	22.0 ± 5.3
Week 6	n = 10/10/10	n = 10/10/10	n = 10/10/10	n = 10/9/10	n = 10/7/10
TSH [ng/L]
male	0.71 ± 0.14	0.72 ± 0.19	0.86 ± 0.31	1.56 ± 0.81**	2.13 ± 0.90**
female	0.52 ± 0.12	0.52 ± 0.11	0.48 ± 0.11	0.82 ± 0.20**	1.35 ± 0.43**
T3 [nmol/L]
male	0.74 ± 0.25	0.63 ± 0.20	0.71 ± 0.24	0.85 ± 0.22	0.71 ± 0.19
female	0.77 ± 0.15	0.72 ± 0.17	0.85 ± 0.18	0.96 ± 0.23	1.16 ± 0.34**
T4 [nmol/L]
male	39.3 ± 7.8	37.7 ± 2.9	37.6 ± 4.1	30.5 ± 4.0**	21.3 ± 3.1**
female	31.4 ± 6.4	34.6 ± 4.2	32.9 ± 5.7	24.1 ± 3.8	23.6 ± 4.0**
Week 13	n = 9/8/10	n = 9/8/10	n = 9/9/10	n = 9/6/10	n = 3/10/10
TSH [ng/L]
male	0.83 ± 0.35	0.80 ± 0.16	0.74 ± 0.10	1.50 ± 0.41	1.63 ± 0.25
female	0.64 ± 0.20	0.64 ± 0.17	0.62 ± 0.10	0.81 ± 0.19	1.44 ± 0.72**
T3 [nmol/L]
male	0.66 ± 0.19	0.75 ± 0.19	0.66 ± 0.15	0.83 ± 0.38	1.09 ± 0.24*
female	0.73 ± 0.11	0.76 ± 0.23	0.91 ± 0.20	1.08 ± 0.18	1.26 ± 0.35**
T4 [nmol/L]
male	36.8 ± 4.6	36.3 ± 2.8	37.5 ± 6.8	29.8 ± 3.0*	22.2 ± 3.4**
female	23.5 ± 3.6	26.3 ± 8.3	29.8 ± 5.8	22.1 ± 4.2	22.9 ± 3.9

TSH = thyroid stimulating hormon; T3 = triiodothyronine; T4 = thyroxin; SD = standard deviation

n = x/y/z = number of males/females/total animals per group

The mean value of the test group was significantly different from control with Dunnett's or Mann-Whitney's test of significance at significance levels of * α = 0.05 and ** α = 0.01.

Table 5. Microscopic liver changes in treated rats when compared with controls

	male		female
Dose (ppm)	500	2 500	500	2 500
Hepatocyte hypertrophy -generalized (severe)                                               -total Hepatocyte hypertrophy -centrilobular becominggeneralized     -mild - moderate - marked - severe - total   Hepatocyte hypertrophy -centrilobular - mild - moderate - total   Cytoplasmic golden brown pigment - mild -moderate - total	0	0	2	4
	I	0	0	0
	I	0	2	0
	0	0	3	3
	0	0	0	3
	2	0	5	6
	5	0	0	0
	I	2	0	0
	6	2	0	0
	0	2	2	4
	0	0	0	4
	0	2	2	8
total number of livers examined	9	2	7	10

 

Table 6. Microscopic thyroid changes in treated rats when compared with controls

	male				female
Dose (ppm)	5	20	500	2500	5	20	500	2 500
Follicular epithelial hypertrophy / hyperplasia - slight - mild - moderate - marked   -total	1	1	0	0	0	0	0	0
	0	2	5	0	0	0	6	3
	0	0	3	2	0	0	0	6
	0	0	1	0	0	0	0	1
	1	3	9	2	0	0	6	10
total number of thyroids examined	IO	10	9	2	9	10	7	10

 

Conclusions:

Sub-chronic dietary administration of the test substance to rats resulted in mortality, decreased red blood cell parameters, changes in clinical pathology parameters, increased liver weights together with hepatocyte hypertrophy, and increased thyroid weights together with follicular hypertrophy/hyperplasia and changes in thyroid hormone levels. Based on these findings, the No Observed Adverse Effect Level (NOAEL) was found to be 20 ppm, approximately 1.2 and 1.5 mg/kg bw/day in males and females, respectively.

Reference 3

Endpoint:

chronic toxicity: 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:

other: 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

Limit test:

no

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 oral 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

Dose / conc.:

5 ppm

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

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

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

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

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

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

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

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 “HISTOPATHOLOGICAL FINDINGS: 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.
(for more details please see Table 4 under “Any other information of results incl. tables”)

- 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.

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)

For more information, please see Section .7.7 Carcinogenicity, key, 2002, M-203024-02-1, RL2, Carcinogenicity (rat, 104 weeks)

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.

Key result

Dose descriptor:

NOAEL

Remarks:

Toxicity

Effect level:

20 ppm

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)

Key result

Dose descriptor:

LOAEL

Remarks:

Toxicity

Effect level:

75 ppm

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: 3.73 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

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 Biliruben 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. 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 52 (Chronic Phase)

Finding Dose Level (ppm in diet) Dose Level (ppm in diet) 0 5 20 75 250 0 5 20 75 250 Males Females Liver                    number examined 10 10 10 10 9 10 10 10 10 10 Hepatocyte hypertrophy - centrilobular 0 1 0 0 0 0 0 1 0 10 Thyroid                number examined 10 10 10 10 9 10 10 10 10 10 Follicular cell hypertrophy 0 0 1 3 5 0 0 2 3 9

 

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.
The substance induced an imbalance of thyroid hormones resulting in higher levels of TSH and lower levels of T4. 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 and carcinogenicity phases in both sexes at 250 ppm. 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. Both hepatocellular hypertrophy and follicular cell hypertrophy, were completely reversible in females during a 13-week recovery period. Follicular cell hypertrophy was still present in the thyroid of males following the 13-week 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.
The No Observed Adverse Effect level (NOAEL) with regard to target organ toxicity was 20 ppm corresponding to 0.98 and 1.34 mg/kg bw/day in males and females, respectively.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

0.98 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

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Oct 1999 - 05 Nov 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)

Version / remarks:

1998

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Version / remarks:

Current version adopted in 1981

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Albino Rats (Outbred) VAF/Plus® CD® (Sprague-Dawley derived) [Crl:CD®(SD)IGS BR]

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Kingston, USA
- Age at study initiation: 8 weeks
- Weight at study initiation: 231–284 g (males) and 183–218 g (females)
- Housing: animals were housed individually in suspended, stainless steel wire mesh cages.
- Diet: certified Rodent Diet No. 5002 (PMI Feeds, Inc., St. Louis, Missouri), pelleted, ad libitum
- Water: water was available via an automated watering system (Elizabethtown Water Company, Westfield, USA).
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-28
- Humidity (%): 36-78
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST SITE
- Area of exposure: dorsal area
- % coverage: 10
- Type of wrap if used: the test substance was evenly spread over a saline-moistened gauze patch, which was placed on the skin and secured with gauze and Elastoplast®.
- Time intervals for shavings or clippings: approximately 24 h before initiation of dosing

REMOVAL OF TEST SUBSTANCE
- Washing: water and gauze were used to remove residual test substance.
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount(s) applied:
10 mg/kg bw/day: 2.62-3.76 mg (m) and 2.03-2.55 mg (f)
20 mg/kg bw/day: 5.24-7.52 mg (m) and 4.06-5.1 mg (f)
500 mg/kg bw/day: 131-188 mg (m) and 101.5-127.5 mg (f)
2500 mg/kg bw/day: 655-940 mg (m) and 507.5-637.5 mg (f)
- Constant volume or concentration used: no
- For solids, paste formed: no

USE OF RESTRAINERS FOR PREVENTING INGESTION: no

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

at least 28 days

Frequency of treatment:

daily, 7 days/week

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, sham-exposed

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were observed, in their cages, for mortality and general appearance twice daily, once in the morning and once in the afternoon. Observations for signs of toxic or pharmacologic effects were made once daily. Any abnormal signs were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: each animal was removed from its cage and examined twice pre-test and once weekly during the study period.
- Clinical observations included: observations of general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia, occurrence of secretions and excretions, and autonomic activity, changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypy or bizarre behaviour

DERMAL IRRITATION: Yes
- Time schedule for examinations: the treated skin was evaluated for irritation and scored pre-test, and prior to dosing for the first week (days 0, 1, 2, 3, 4, 5 and 6) and weekly thereafter during the treatment period and again just prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed twice pre-test, weekly during the study and at termination.

FOOD CONSUMPTION:
- Food consumption for each group determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: ophthalmoscopic examinations were performed at Week -1 (pre-test) and Week 4.
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: blood samples were obtained at study termination (Week 5) via retrobulbar venous plexus.
- Anaesthetic used for blood collection: Yes (light CO2/O2)
- Animals fasted: Yes
- How many animals: 100
- Parameters listed in Table 1 under “Any other information on materials and methods incl. tables” were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: blood samples were obtained from the aorta at necropsy (Week 5).
- Animals fasted: Yes
- How many animals: 100
- Parameters listed in Table 1 under “Any other information on materials and methods incl. tables” were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: at Weeks 4 and 5
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (at Week 5)
- Parameters listed in Table 1 under “Any other information on materials and methods incl. tables” were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: pre-test and at Week 4
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Table 2 under “Any other information on materials and methods incl. tables”)
HISTOPATHOLOGY: Yes (see Table 2 under “Any other information on materials and methods incl. tables”)

Statistics:

The mean values of the following parameters comparing control and test substance-treated groups were analysed statistically: body weight, food consumption, terminal organ weights, organ/body and organ/brain weight ratios, clinical pathology and motor activity counts. Bartlett's test was performed to determine if groups had equal variances. If the variances were equal, parametric procedures were used; if not, nonparametric procedures were used. The parametric method was the standard one-way analysis of variance (ANOVA) using the F ratio to assess significance followed by Dunnett's or Williams test to determine which means were significantly different from the control. The nonparametric method was the Kruskal-Wallis test followed by Shirley's or Dunn's test or pairwise comparison with Bonferroni correction to determine which mean values significantly differed from control.

Clinical signs:

no effects observed

Description (incidence and severity):

All test and control animals survived and were free of significant clinical signs throughout the study.

Dermal irritation:

no effects observed

Mortality:

no mortality observed

Description (incidence):

All test and control animals survived until the end of the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weight values for the test groups were comparable to the control values throughout the study. At Week 1, mean body weight and body weight gain were statistically significantly decreased (-7.4%) in males of the 1000 mg/kg bw/day group compared to controls. Since body weight gain was also statistically significantly decreased in the pre-test period, as compared to the control value, the body weight effects in Week 1 could not be attributed solely to the test substance. Mean body weights for the 1000 mg/kg bw/day males during the remainder of the study were slightly lower than those of the control males, although the decreases were not statistically significant. In females, a slight, but statistically significant, increase in body weight gain in Week 1 was observed for the 250 and 1000 mg/kg bw/day groups, as compared to the controls. In subsequent weeks, body weight gains for these animals were comparable to the control values.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean food consumption values for the test groups were generally comparable to, or slightly greater than, the control values throughout the study. At Week 1, the mean feed consumption values for the 1000 mg/kg bw/day males and females were statistically significantly decreased compared to the controls. In subsequent weeks, food consumption values of these animals were comparable to the control values.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There was no evidence for test substance-related ocular abnormalities at termination of the study.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Mean haemoglobin concentration, haematocrit and erythrocyte counts were statistically significantly decreased for males and females at 250 and 1000 mg/kg bw/day. In females, haematocrit was also statistically significantly decreased at 50 mg/kg bw/day. The decreases appeared to be test substance-related, although the decreases were slight (less than 10%) and no compensatory increase in reticulocyte counts was noted at any dose level. Activated partial thromboplastin times were significantly and dose-dependently prolonged in males and females at 50, 250, and 1000 mg/kg bw/day. Statistically significant increases in prothrombin time were observed at 250 and 1000 mg/kg/day in males and at 1000 mg/kg bw/day in females. Platelet counts were generally increased in males and females at 50, 250 and 1000 mg/kg bw/day. However, only the increases in males at 50 mg/kg bw/day and in females at 250 and 1000 mg/kg bw/day were statistically significant. A few statistically significant decreases in the counts of leukocytes, absolute lymphocytes and eosinophils were noted at the high-dose group (1000 mg/kg bw/day) in females, but these slight changes were not considered to be biologically significant.
(see Table 3 under “Any other information on results incl. tables”)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related changes in clinical chemistry parameters were observed in males and females at 250 and 1000 mg/kg/day (see Table 3 under “Any other information on results incl. tables”). In general, the affected parameters were those usually related to hepatic changes (e.g. gamma-glutamyl transferase, alanine aminotransferase, cholesterol). Only a few statistically significant changes in clinical chemistry parameters were noted at 50 mg/kg bw/day (decreased albumin/globulin ratio in males and decreased globulin values in females).

Urinalysis findings:

no effects observed

Description (incidence and severity):

The urinalysis values of the test animals were comparable to control animal values, or within the range of normal variability.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No test substance-related effects on motor activity and no indications of neurological effects were observed at any dose group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

≥ 50 mg/kg bw/day: increased abs. and rel. liver weight and liver/brain weight ratios; ≥ 250 mg/kg bw/day (f): increased abs. and rel. adrenal gland weight and adrenal/brain weight ratios

The mean liver weight and the mean liver/body weight and liver/brain weight ratios for the 50, 250, and 1000 mg/kg bw/day males and females were statistically significantly increased compared to control group values. The increases in liver weight values were dose-related and are consistent with the observed changes in clinical chemistry parameters indicative of hepatotoxicity. The mean adrenal weight and the mean adrenal/body weight and adrenal/brain weight ratios for the 250 and 1000 mg/kg bw/day females were also statistically significantly increased, as compared to control female values. These increases are consistent with histopathological changes in the adrenal cortex of females treated with the high-dose (1000 mg/kg bw/day).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

≥ 50 mg/kg bw/day: brown discolouration and scattered tan foci in liver

A test article-related brown discolouration and scattered tan foci were found in males and females at 50, 250 and 1000 mg/kg bw/day. Other macroscopic findings involved sporadic incidences in control and test substance treated groups. These incidental findings were not considered to be test substance-related, since they were also observed in rats of this strain and age used in previous studies.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

≥ 50 mg/kg bw/day: centrilobular hepatocellular hypertrophy and follicular cell hyperthrophy/hyperplasia in thyroid gland; ≥ 250 mg/kg bw/day (f): hypertrophy/hyperplasia in adrenal cortex (zona fasiculata)

Centrilobular hepatocellular hypertrophy was present in all males and females at 250 and 1000 mg/kg bw/day, and in males (3/10 ) and females (6/10) at 50 mg/kg bw/day. Follicular cell hypertrophy/hyperplasia in the thyroid gland was present in all males and females at 250 and 1000 mg/kg bw/day and in males (4/10) and females (3/10) at 50 mg/kg bw/day. In the adrenal cortex, hypertrophy/hyperplasia of the zona fasiculata was present in females at 250 mg/kg bw/day (1/10) and females at 1000 mg/kg bw/day (5/10). In liver, thyroid and adrenal gland, a dose-related increase in severity of histopathological effects was observed.
(see Table 4 under “Any other information on results incl. tables”)

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The skin at the site of the topical application of either the vehicle or the vehicle/test article combination had squamous cell hyperplasia, hyperkeratosis and/or hyperplasia of parafollicular sebaceous glands; all findings were of minimal severity. The liver of a number of rats, including controls, had lesions possibly associated with wrapping of the torso following dermal application of the test article (Parker and Gibson, 1995). These lesions were focal/multifocal, subcapsular, in various stages of development and minimal to slight in severity. The acute lesions consisted of coagulative necrosis, occasional haemorrhage and acute/subacute inflammation. Older lesions consisted of subacute/chronic inflammation and various stages of fibroplasia leading to focal fibrosis. These findings in the skin and liver were considered to be procedurally related rather than vehicle or test article-related. Other microscopic findings in the liver, thyroid and adrenal glands and in the other tissues and organs occurred with comparable incidence and severity in rats from the control and test article treatment groups. These incidental findings, not considered to be test article related, have been seen in rats of this strain and age used in other studies conducted in this facility.

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Key result

Dose descriptor:

LOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day (nominal)

System:

other: hepatobiliary and endocrine system

Organ:

liver

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Table 3. Changes in clinical chemistry parameters (data are presented as mean values ± SD)

Parameters	Test substance [mg/kg bw/day]
	0	10	50	250	1000
HGB [g/dL]
male	16 ± 0.4	14.6 ± 3.4	15.8 ± 0.4	15.3 ± 0.5*	14.8 ± 0.6**
female	15.5 ± 0.4	15.2 ± 0.5	14.9 ± 0.7	14.1 ± 0.7**	14.2 ± 0.5**
HCT [%]
male	47.7 ± 1.2	46.9 ± 1.1	47.5 ± 1.4	45.4 ± 1.8**	44.7 ± 2.1**
female	45.9 ± 1.4	44.9 ± 1.7	43.6 ± 1.8*	42.1 ± 2.0**	42.2 ± 1.6**
RBC [10E+6/µL]
male	8.64 ± 0.29	8.56 ± 0.36	8.50 ± 0.30	8.23 ± 0.43*	8.21 ± 0.40*
female	8.30 ± 0.35	8.07 ± 0.31	7.94 ± 0.47	7.65 ± 0.31**	7.68 ± 0.36**
PLT [10E+3/µL]
male	770 ± 183	763 ± 214	1017 ± 125*	922 ± 235	937 ± 256
female	821 ± 333	847 ± 282	950 ± 287	1312 ± 263**	1255 ± 260**
WBC [10E+3/µL]
male	11.4 ± 2.9	12.9 ± 2.5	10.8 ± 2.7	9.9 ± 2.6	9.2 ± 1.5
female	12.7 ± 3.1	12.8 ± 3.1	10.6 ± 2.9	9.6 ± 2.3	8.8 ± 2.6*
PT [s]
male	12.6 ± 0.8	12.5 ± 0.5	14.2 ± 1.5	18.8 ± 4.3*	21.4 ± 4.9**
female	11.2 ± 0.4	11.2 ± 0.3	11.4 ± 0.5	11.8 ± 0.7	13.8 ± 1.9**
APTT [s]
male	18.9 ± 2.4	19.1 ± 1.5	22.2 ± 2.1*	25.7 ± 3.8**	27.9 ± 3.1**
female	13.9 ± 2.2	15.3 ± 1.1	17.2 ± 3.1*	19.8 ± 2.5**	22.8 ± 3.1**
ANEU [10E+3/µL]
male	1.46 ± 0.38	1.47 ± 0.24	1.28 ± 0.31	1.34 ± 0.37	1.02 ± 0.27*
female	1.54 ± 0.60	1.24 ± 0.67	1.75 ± 0.98	1.26 ± 0.65	1.04 ± 0.41
ALYM [10E+3/µL]
male	9.41 ± 2.78	10.89 ± 2.44	9.00 ± 2.77	7.98 ± 2.54	7.85 ± 1.40
female	10.48 ± 2.66	11.02 ± 2.80	8.33 ± 1.98	7.95 ± 1.83	7.39 ± 2.69*
EOS [% WBC]
male	1.8 ± 0.9	1.5 ± 0.9	1.5 ± 1.5	2.7 ± 2.2	1.4 ± 1.2
female	2.0 ± 1.6	1.6 ± 1.0	1.1 ± 0.9	1.0 ± 0.8	0.5 ± 0.3**
GGT [IU/L]
male	0 ± 0	1 ± 1	1 ± 1	3 ± 1**	10 ± 3**
female	1 ± 1	1 ± 1	2 ± 2	7 ± 2**	15 ± 5**
TP [g/dL]
male	6.1 ± 0.2	6.0 ± 0.4	6.3 ± 0.6	6.6 ± 0.3*	6.9 ± 0.4**
female	6.2 ± 0.5	6.3 ± 0.4	6.6 ± 0.3	7.1 ± 0.4**	7.4 ± 0.6**
GLOB [g/dL]
male	1.8 ± 0.2	1.8 ± 0.2	2.1 ± 0.3	2.4 ± 0.2**	2.5 ± 0.3**
female	1.8 ± 0.4	1.8 ± 0.2	2.2 ± 0.3*	2.5 ± 0.4**	2.8 ± 0.3**
A/G
male	2.3 ± 0.3	2.3 ± 0.3	2.1 ± 0.2*	1.8 ± 0.2**	1.8 ± 0.2**
female	2.6 ± 0.6	2.6 ± 0.3	2.1 ± 0.4	1.9 ± 0.4**	1.6 ± 0.3**
CHOL [mg/dL]
male	41 ± 017	47 ± 13	41 ± 12	57 ± 9	64 ± 18**
female	53 ± 11	50 ± 15	58 ± 12	84 ± 20*	117 ± 40**
GLUC [mg/dL]
male	154 ± 26	143 ± 28	138 ± 23	121 ± 16**	112 ± 12**
female	138 ± 23	142 ± 29	147 ± 29	146 ± 26	130 ± 24
ALT [IU/L]
male	41 ± 10	41 ± 14	52 ± 22	50 ± 13	75 ± 25**
female	35 ± 11	58 ± 91	90 ± 169	50 ± 24	50 ± 17
ALKP [IU/L]
male	162 ± 25	169 ± 21	166 ± 23	153 ± 29	124 ± 25**
female	98 ± 23	92 ± 11	82 ± 13	68 ± 15**	71 ± 21**
Cl- [mEq/L]
male	100 ± 2	101 ± 2	101 ± 2	100 ± 1	101 ± 1
female	104 ± 2	103 ± 2	103 ± 1	102 ± 2	101 ± 3*

HGB = haemoglobin; HCT = haematocrit; RBC = erythrocyte count; PLT = platelet count; WBC = total leukocyte count; PT = prothrombin time; APTT = activated partial thromboplastin time; ANEU = absolute neutrophils; ALYM = absolute lymphocytes; EOS = eosinophil count; GGT = gamma-glutamyl transferase; TP = total protein; GLOB = globulin; A/G = albumin/globulin ratio; CHOL = cholesterol; GLUC = glucose; ALT = alanine aminotransferase; ALKP = alkaline phosphatase; SD = standard deviation

Statistical significance is indicated by *p < 0.05, **p < 0.01 and ***p < 0.001.

Table 4. Histopathological changes in liver, thyroid and adrenal gland

Liver: hepatocellular hypertrophy (centrilobular)
	Males					Females
Dose [mg/kg bw/day]	0	10	50	250	1000	0	10	50	250	1000
Number examined	10	10	10	10	10	10	10	10	10	10
Severity
No abnormal diagnosis	10	10	7	0	0	10	10	4	0	0
Minimal	0	0	3	0	0	0	0	5	0	0
Slight	0	0	0	9	0	0	0	1	5	3
Moderate	0	0	0	1	10	0	0	0	5	7
Total findings	0	0	3	10	10	0	0	6	10	10
Thyroid gland: follicular cell hypertrophy/hyperplasia
	Males					Females
Dose [mg/kg bw/day]	0	10	50	250	1000	0	10	50	250	1000
Number examined	10	9	10	10	10	10	10	7	0	0
Severity
No abnormal diagnosis	10	9	6	0	0	10	10	7	0	0
Minimal	0	0	4	0	0	0	0	3	0	0
Slight	0	0	0	5	0	0	0	0	5	1
Moderate	0	0	0	5	10	0	0	0	5	9
Total findings	0	0	4	10	10	0	0	3	10	10
Adrenal cortex: zona fasiculata-hypertrophy/hyperplasia
	Males					Females
Dose [mg/kg bw/day]	0	10	50	250	1000	0	10	50	250	1000
Number examined	10	0	0	0	10	10	10	10	10	10
Severity
No abnormal diagnosis	10	-	-	-	10	10	10	10	9	5
Slight	0	-	-	-	0	0	0	0	1	5
Total findings	0	-	-	-	10	0	0	0	1	5

Conclusions:

Sub-acute dermal administration of the test substance to rats resulted in increase in liver weight, the presence of centrilobular hepatocellular hypertrophy and follicular cell hypertrophy/hyperplasia in thyroid glands as well as changes in clinical chemistry parameters indicative of hepatotoxicity at 50 mg/kg bw/day. Based on these findings, the No Observed Adverse Effect Level (NOAEL) was found to be 10 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study, 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

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Oct 1999 - 05 Nov 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)

Version / remarks:

1998

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Version / remarks:

Current version adopted in 1981

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Albino Rats (Outbred) VAF/Plus® CD® (Sprague-Dawley derived) [Crl:CD®(SD)IGS BR]

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Kingston, USA
- Age at study initiation: 8 weeks
- Weight at study initiation: 231–284 g (males) and 183–218 g (females)
- Housing: animals were housed individually in suspended, stainless steel wire mesh cages.
- Diet: certified Rodent Diet No. 5002 (PMI Feeds, Inc., St. Louis, Missouri), pelleted, ad libitum
- Water: water was available via an automated watering system (Elizabethtown Water Company, Westfield, USA).
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-28
- Humidity (%): 36-78
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST SITE
- Area of exposure: dorsal area
- % coverage: 10
- Type of wrap if used: the test substance was evenly spread over a saline-moistened gauze patch, which was placed on the skin and secured with gauze and Elastoplast®.
- Time intervals for shavings or clippings: approximately 24 h before initiation of dosing

REMOVAL OF TEST SUBSTANCE
- Washing: water and gauze were used to remove residual test substance.
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount(s) applied:
10 mg/kg bw/day: 2.62-3.76 mg (m) and 2.03-2.55 mg (f)
20 mg/kg bw/day: 5.24-7.52 mg (m) and 4.06-5.1 mg (f)
500 mg/kg bw/day: 131-188 mg (m) and 101.5-127.5 mg (f)
2500 mg/kg bw/day: 655-940 mg (m) and 507.5-637.5 mg (f)
- Constant volume or concentration used: no
- For solids, paste formed: no

USE OF RESTRAINERS FOR PREVENTING INGESTION: no

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

at least 28 days

Frequency of treatment:

daily, 7 days/week

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, sham-exposed

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were observed, in their cages, for mortality and general appearance twice daily, once in the morning and once in the afternoon. Observations for signs of toxic or pharmacologic effects were made once daily. Any abnormal signs were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: each animal was removed from its cage and examined twice pre-test and once weekly during the study period.
- Clinical observations included: observations of general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia, occurrence of secretions and excretions, and autonomic activity, changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypy or bizarre behaviour

DERMAL IRRITATION: Yes
- Time schedule for examinations: the treated skin was evaluated for irritation and scored pre-test, and prior to dosing for the first week (days 0, 1, 2, 3, 4, 5 and 6) and weekly thereafter during the treatment period and again just prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed twice pre-test, weekly during the study and at termination.

FOOD CONSUMPTION:
- Food consumption for each group determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: ophthalmoscopic examinations were performed at Week -1 (pre-test) and Week 4.
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: blood samples were obtained at study termination (Week 5) via retrobulbar venous plexus.
- Anaesthetic used for blood collection: Yes (light CO2/O2)
- Animals fasted: Yes
- How many animals: 100
- Parameters listed in Table 1 under “Any other information on materials and methods incl. tables” were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: blood samples were obtained from the aorta at necropsy (Week 5).
- Animals fasted: Yes
- How many animals: 100
- Parameters listed in Table 1 under “Any other information on materials and methods incl. tables” were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: at Weeks 4 and 5
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (at Week 5)
- Parameters listed in Table 1 under “Any other information on materials and methods incl. tables” were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: pre-test and at Week 4
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Table 2 under “Any other information on materials and methods incl. tables”)
HISTOPATHOLOGY: Yes (see Table 2 under “Any other information on materials and methods incl. tables”)

Statistics:

The mean values of the following parameters comparing control and test substance-treated groups were analysed statistically: body weight, food consumption, terminal organ weights, organ/body and organ/brain weight ratios, clinical pathology and motor activity counts. Bartlett's test was performed to determine if groups had equal variances. If the variances were equal, parametric procedures were used; if not, nonparametric procedures were used. The parametric method was the standard one-way analysis of variance (ANOVA) using the F ratio to assess significance followed by Dunnett's or Williams test to determine which means were significantly different from the control. The nonparametric method was the Kruskal-Wallis test followed by Shirley's or Dunn's test or pairwise comparison with Bonferroni correction to determine which mean values significantly differed from control.

Clinical signs:

no effects observed

Description (incidence and severity):

All test and control animals survived and were free of significant clinical signs throughout the study.

Dermal irritation:

no effects observed

Mortality:

no mortality observed

Description (incidence):

All test and control animals survived until the end of the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weight values for the test groups were comparable to the control values throughout the study. At Week 1, mean body weight and body weight gain were statistically significantly decreased (-7.4%) in males of the 1000 mg/kg bw/day group compared to controls. Since body weight gain was also statistically significantly decreased in the pre-test period, as compared to the control value, the body weight effects in Week 1 could not be attributed solely to the test substance. Mean body weights for the 1000 mg/kg bw/day males during the remainder of the study were slightly lower than those of the control males, although the decreases were not statistically significant. In females, a slight, but statistically significant, increase in body weight gain in Week 1 was observed for the 250 and 1000 mg/kg bw/day groups, as compared to the controls. In subsequent weeks, body weight gains for these animals were comparable to the control values.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean food consumption values for the test groups were generally comparable to, or slightly greater than, the control values throughout the study. At Week 1, the mean feed consumption values for the 1000 mg/kg bw/day males and females were statistically significantly decreased compared to the controls. In subsequent weeks, food consumption values of these animals were comparable to the control values.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There was no evidence for test substance-related ocular abnormalities at termination of the study.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Mean haemoglobin concentration, haematocrit and erythrocyte counts were statistically significantly decreased for males and females at 250 and 1000 mg/kg bw/day. In females, haematocrit was also statistically significantly decreased at 50 mg/kg bw/day. The decreases appeared to be test substance-related, although the decreases were slight (less than 10%) and no compensatory increase in reticulocyte counts was noted at any dose level. Activated partial thromboplastin times were significantly and dose-dependently prolonged in males and females at 50, 250, and 1000 mg/kg bw/day. Statistically significant increases in prothrombin time were observed at 250 and 1000 mg/kg/day in males and at 1000 mg/kg bw/day in females. Platelet counts were generally increased in males and females at 50, 250 and 1000 mg/kg bw/day. However, only the increases in males at 50 mg/kg bw/day and in females at 250 and 1000 mg/kg bw/day were statistically significant. A few statistically significant decreases in the counts of leukocytes, absolute lymphocytes and eosinophils were noted at the high-dose group (1000 mg/kg bw/day) in females, but these slight changes were not considered to be biologically significant.
(see Table 3 under “Any other information on results incl. tables”)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related changes in clinical chemistry parameters were observed in males and females at 250 and 1000 mg/kg/day (see Table 3 under “Any other information on results incl. tables”). In general, the affected parameters were those usually related to hepatic changes (e.g. gamma-glutamyl transferase, alanine aminotransferase, cholesterol). Only a few statistically significant changes in clinical chemistry parameters were noted at 50 mg/kg bw/day (decreased albumin/globulin ratio in males and decreased globulin values in females).

Urinalysis findings:

no effects observed

Description (incidence and severity):

The urinalysis values of the test animals were comparable to control animal values, or within the range of normal variability.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No test substance-related effects on motor activity and no indications of neurological effects were observed at any dose group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

≥ 50 mg/kg bw/day: increased abs. and rel. liver weight and liver/brain weight ratios; ≥ 250 mg/kg bw/day (f): increased abs. and rel. adrenal gland weight and adrenal/brain weight ratios

The mean liver weight and the mean liver/body weight and liver/brain weight ratios for the 50, 250, and 1000 mg/kg bw/day males and females were statistically significantly increased compared to control group values. The increases in liver weight values were dose-related and are consistent with the observed changes in clinical chemistry parameters indicative of hepatotoxicity. The mean adrenal weight and the mean adrenal/body weight and adrenal/brain weight ratios for the 250 and 1000 mg/kg bw/day females were also statistically significantly increased, as compared to control female values. These increases are consistent with histopathological changes in the adrenal cortex of females treated with the high-dose (1000 mg/kg bw/day).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

≥ 50 mg/kg bw/day: brown discolouration and scattered tan foci in liver

A test article-related brown discolouration and scattered tan foci were found in males and females at 50, 250 and 1000 mg/kg bw/day. Other macroscopic findings involved sporadic incidences in control and test substance treated groups. These incidental findings were not considered to be test substance-related, since they were also observed in rats of this strain and age used in previous studies.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

≥ 50 mg/kg bw/day: centrilobular hepatocellular hypertrophy and follicular cell hyperthrophy/hyperplasia in thyroid gland; ≥ 250 mg/kg bw/day (f): hypertrophy/hyperplasia in adrenal cortex (zona fasiculata)

Centrilobular hepatocellular hypertrophy was present in all males and females at 250 and 1000 mg/kg bw/day, and in males (3/10 ) and females (6/10) at 50 mg/kg bw/day. Follicular cell hypertrophy/hyperplasia in the thyroid gland was present in all males and females at 250 and 1000 mg/kg bw/day and in males (4/10) and females (3/10) at 50 mg/kg bw/day. In the adrenal cortex, hypertrophy/hyperplasia of the zona fasiculata was present in females at 250 mg/kg bw/day (1/10) and females at 1000 mg/kg bw/day (5/10). In liver, thyroid and adrenal gland, a dose-related increase in severity of histopathological effects was observed.
(see Table 4 under “Any other information on results incl. tables”)

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The skin at the site of the topical application of either the vehicle or the vehicle/test article combination had squamous cell hyperplasia, hyperkeratosis and/or hyperplasia of parafollicular sebaceous glands; all findings were of minimal severity. The liver of a number of rats, including controls, had lesions possibly associated with wrapping of the torso following dermal application of the test article (Parker and Gibson, 1995). These lesions were focal/multifocal, subcapsular, in various stages of development and minimal to slight in severity. The acute lesions consisted of coagulative necrosis, occasional haemorrhage and acute/subacute inflammation. Older lesions consisted of subacute/chronic inflammation and various stages of fibroplasia leading to focal fibrosis. These findings in the skin and liver were considered to be procedurally related rather than vehicle or test article-related. Other microscopic findings in the liver, thyroid and adrenal glands and in the other tissues and organs occurred with comparable incidence and severity in rats from the control and test article treatment groups. These incidental findings, not considered to be test article related, have been seen in rats of this strain and age used in other studies conducted in this facility.

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Key result

Dose descriptor:

LOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day (nominal)

System:

other: hepatobiliary and endocrine system

Organ:

liver

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Table 3. Changes in clinical chemistry parameters (data are presented as mean values ± SD)

Parameters	Test substance [mg/kg bw/day]
	0	10	50	250	1000
HGB [g/dL]
male	16 ± 0.4	14.6 ± 3.4	15.8 ± 0.4	15.3 ± 0.5*	14.8 ± 0.6**
female	15.5 ± 0.4	15.2 ± 0.5	14.9 ± 0.7	14.1 ± 0.7**	14.2 ± 0.5**
HCT [%]
male	47.7 ± 1.2	46.9 ± 1.1	47.5 ± 1.4	45.4 ± 1.8**	44.7 ± 2.1**
female	45.9 ± 1.4	44.9 ± 1.7	43.6 ± 1.8*	42.1 ± 2.0**	42.2 ± 1.6**
RBC [10E+6/µL]
male	8.64 ± 0.29	8.56 ± 0.36	8.50 ± 0.30	8.23 ± 0.43*	8.21 ± 0.40*
female	8.30 ± 0.35	8.07 ± 0.31	7.94 ± 0.47	7.65 ± 0.31**	7.68 ± 0.36**
PLT [10E+3/µL]
male	770 ± 183	763 ± 214	1017 ± 125*	922 ± 235	937 ± 256
female	821 ± 333	847 ± 282	950 ± 287	1312 ± 263**	1255 ± 260**
WBC [10E+3/µL]
male	11.4 ± 2.9	12.9 ± 2.5	10.8 ± 2.7	9.9 ± 2.6	9.2 ± 1.5
female	12.7 ± 3.1	12.8 ± 3.1	10.6 ± 2.9	9.6 ± 2.3	8.8 ± 2.6*
PT [s]
male	12.6 ± 0.8	12.5 ± 0.5	14.2 ± 1.5	18.8 ± 4.3*	21.4 ± 4.9**
female	11.2 ± 0.4	11.2 ± 0.3	11.4 ± 0.5	11.8 ± 0.7	13.8 ± 1.9**
APTT [s]
male	18.9 ± 2.4	19.1 ± 1.5	22.2 ± 2.1*	25.7 ± 3.8**	27.9 ± 3.1**
female	13.9 ± 2.2	15.3 ± 1.1	17.2 ± 3.1*	19.8 ± 2.5**	22.8 ± 3.1**
ANEU [10E+3/µL]
male	1.46 ± 0.38	1.47 ± 0.24	1.28 ± 0.31	1.34 ± 0.37	1.02 ± 0.27*
female	1.54 ± 0.60	1.24 ± 0.67	1.75 ± 0.98	1.26 ± 0.65	1.04 ± 0.41
ALYM [10E+3/µL]
male	9.41 ± 2.78	10.89 ± 2.44	9.00 ± 2.77	7.98 ± 2.54	7.85 ± 1.40
female	10.48 ± 2.66	11.02 ± 2.80	8.33 ± 1.98	7.95 ± 1.83	7.39 ± 2.69*
EOS [% WBC]
male	1.8 ± 0.9	1.5 ± 0.9	1.5 ± 1.5	2.7 ± 2.2	1.4 ± 1.2
female	2.0 ± 1.6	1.6 ± 1.0	1.1 ± 0.9	1.0 ± 0.8	0.5 ± 0.3**
GGT [IU/L]
male	0 ± 0	1 ± 1	1 ± 1	3 ± 1**	10 ± 3**
female	1 ± 1	1 ± 1	2 ± 2	7 ± 2**	15 ± 5**
TP [g/dL]
male	6.1 ± 0.2	6.0 ± 0.4	6.3 ± 0.6	6.6 ± 0.3*	6.9 ± 0.4**
female	6.2 ± 0.5	6.3 ± 0.4	6.6 ± 0.3	7.1 ± 0.4**	7.4 ± 0.6**
GLOB [g/dL]
male	1.8 ± 0.2	1.8 ± 0.2	2.1 ± 0.3	2.4 ± 0.2**	2.5 ± 0.3**
female	1.8 ± 0.4	1.8 ± 0.2	2.2 ± 0.3*	2.5 ± 0.4**	2.8 ± 0.3**
A/G
male	2.3 ± 0.3	2.3 ± 0.3	2.1 ± 0.2*	1.8 ± 0.2**	1.8 ± 0.2**
female	2.6 ± 0.6	2.6 ± 0.3	2.1 ± 0.4	1.9 ± 0.4**	1.6 ± 0.3**
CHOL [mg/dL]
male	41 ± 017	47 ± 13	41 ± 12	57 ± 9	64 ± 18**
female	53 ± 11	50 ± 15	58 ± 12	84 ± 20*	117 ± 40**
GLUC [mg/dL]
male	154 ± 26	143 ± 28	138 ± 23	121 ± 16**	112 ± 12**
female	138 ± 23	142 ± 29	147 ± 29	146 ± 26	130 ± 24
ALT [IU/L]
male	41 ± 10	41 ± 14	52 ± 22	50 ± 13	75 ± 25**
female	35 ± 11	58 ± 91	90 ± 169	50 ± 24	50 ± 17
ALKP [IU/L]
male	162 ± 25	169 ± 21	166 ± 23	153 ± 29	124 ± 25**
female	98 ± 23	92 ± 11	82 ± 13	68 ± 15**	71 ± 21**
Cl- [mEq/L]
male	100 ± 2	101 ± 2	101 ± 2	100 ± 1	101 ± 1
female	104 ± 2	103 ± 2	103 ± 1	102 ± 2	101 ± 3*

HGB = haemoglobin; HCT = haematocrit; RBC = erythrocyte count; PLT = platelet count; WBC = total leukocyte count; PT = prothrombin time; APTT = activated partial thromboplastin time; ANEU = absolute neutrophils; ALYM = absolute lymphocytes; EOS = eosinophil count; GGT = gamma-glutamyl transferase; TP = total protein; GLOB = globulin; A/G = albumin/globulin ratio; CHOL = cholesterol; GLUC = glucose; ALT = alanine aminotransferase; ALKP = alkaline phosphatase; SD = standard deviation

Statistical significance is indicated by *p < 0.05, **p < 0.01 and ***p < 0.001.

Table 4. Histopathological changes in liver, thyroid and adrenal gland

Liver: hepatocellular hypertrophy (centrilobular)
	Males					Females
Dose [mg/kg bw/day]	0	10	50	250	1000	0	10	50	250	1000
Number examined	10	10	10	10	10	10	10	10	10	10
Severity
No abnormal diagnosis	10	10	7	0	0	10	10	4	0	0
Minimal	0	0	3	0	0	0	0	5	0	0
Slight	0	0	0	9	0	0	0	1	5	3
Moderate	0	0	0	1	10	0	0	0	5	7
Total findings	0	0	3	10	10	0	0	6	10	10
Thyroid gland: follicular cell hypertrophy/hyperplasia
	Males					Females
Dose [mg/kg bw/day]	0	10	50	250	1000	0	10	50	250	1000
Number examined	10	9	10	10	10	10	10	7	0	0
Severity
No abnormal diagnosis	10	9	6	0	0	10	10	7	0	0
Minimal	0	0	4	0	0	0	0	3	0	0
Slight	0	0	0	5	0	0	0	0	5	1
Moderate	0	0	0	5	10	0	0	0	5	9
Total findings	0	0	4	10	10	0	0	3	10	10
Adrenal cortex: zona fasiculata-hypertrophy/hyperplasia
	Males					Females
Dose [mg/kg bw/day]	0	10	50	250	1000	0	10	50	250	1000
Number examined	10	0	0	0	10	10	10	10	10	10
Severity
No abnormal diagnosis	10	-	-	-	10	10	10	10	9	5
Slight	0	-	-	-	0	0	0	0	1	5
Total findings	0	-	-	-	10	0	0	0	1	5

Conclusions:

Sub-acute dermal administration of the test substance to rats resulted in increase in liver weight, the presence of centrilobular hepatocellular hypertrophy and follicular cell hypertrophy/hyperplasia in thyroid glands as well as changes in clinical chemistry parameters indicative of hepatotoxicity at 50 mg/kg bw/day. Based on these findings, the No Observed Adverse Effect Level (NOAEL) was found to be 10 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

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

Mode of Action Analysis / Human Relevance Framework

Please refer to the information provided in IUCLID section 13.2.

Additional information

Oral

Subacute

A subacute oral toxicity study was performed in Wistar rats following OECD guideline 407 and in compliance with GLP (M-201261-01-2, 2001a). The test substance was administered ad libitum via the diet to groups of 10 male and 10 female rats at concentrations of 20, 100, 500 and 2500 ppm for 28 days. The corresponding mean dose levels over the study period were 1.8, 9.2, 46.1 and 219.3 mg/kg bw/day in males and 2.0, 9.6, 46.3 and 220.2 mg/kg bw/day in females. A negative control group of 20 animals, consisting of 10 animals per sex, received plain diet.

Three males died during the course of the study. The deaths of these animals were related to treatment in 2 males of the 2500 ppm test group. One male of the 20 ppm test group inadvertently died during anaesthesia for blood sampling. Animals which died at the 2500 ppm group showed a general pallor before death. No treatment-related clinical signs occurred in the surviving animals. In males of the 2500 ppm group, the overall body weight gain was significantly lower compared to controls. A slight reduction in body weight gain in females during the first week of the study was observed at 2500 ppm when compared to controls, which corresponded to a decrease in food consumption at this time point. However, body weights in females treated with the high dose were similar to controls at the end of the study. Food consumption in males at the high dose was statistically significantly decreased during Week 1 and 2 of the study.

There were no treatment-related changes at the ophthalmological and neurobehavioural examinations.

Haematological analysis revealed a significantly higher mean prothrombin time in males at 500 and 2500 ppm and increased mean platelet counts in both genders at 2500 ppm. Some statistically significant changes were also noted in erythrocyte parameters in both sexes. However, as the incidence of abnormal values and/or the amplitude relative to control were low, these changes were not considered to be toxicologically relevant. Alterations in clinical chemistry parameters in blood involved an increase in total cholesterol concentrations in females at ≥ 500 ppm and males treated with the high dose. In females, increased cholesterol values were accompanied by an increase in the triglyceride concentrations at concentrations ≥ 500 ppm. Increased activity of alanine aminotransferase, a parameter indicative of hepatotoxicity, was only observed in males of the high dose group when compared to controls. The concentration of total protein was increased in females at 500 and 2500 ppm. A slight non-toxicologically significant increase in total protein was also observed in males at 2500 ppm and in females at 100 ppm. In males treated with the high dose, a significant decrease in albumin was noted. In the absence of a clear dose-response relationship or biological significance, all other alterations in clinical chemistry parameters (e.g. increased aspartate aminotransferase activity and calcium concentration, decreased glucose and chloride concentration) were judged to be of no toxicological significance. Furthermore, blood samples were analysed for changes in the concentrations of the thyroid hormones T3, T4 and TSH. The results demonstrated a significant increase in TSH levels in both sexes at 500 and 2500 ppm. The increase in TSH hormone levels in males was associated with a decrease in T4 hormone levels at respective doses. Although T3 levels were significantly increased in both males and females of the high dose group, this effect was not considered to be of biological significance, since plasma levels of T3 in the control animals were below the normal physiological range in these rats.

The urinary volume was statistically significantly decreased in males treated with 500 and 2500 ppm when compared to controls. At the high dose, males also showed a slight reduction in urinary pH.

At study termination, treatment- related organ changes were observed in liver, thyroid gland, adrenal gland and kidney.

The absolute and relative organ weights of liver were statistically and dose-relatedly increased in males treated at 500 and 2500 ppm, and in females treated at 100, 500 and 2500 ppm. The increase in liver weight correlated with the macroscopic finding of liver enlargement and the increased incidence and severity of diffuse centrilobular to panlobular hepatocellular hypertrophy in both genders at ≥ 500 ppm. Associated with the enlargement, a dark colour of the liver was observed in males and females at 500 and 2500 ppm. Slight golden brown pigments in the hepatocellular cytoplasm of females treated at 2500 ppm were found at histopathological examination of the liver.

The thyroid gland weights were dose-relatedly and statistically significantly increased in animals treated with 500 and 2500 ppm of the test substance. These changes in organ weights correlated with the increased incidence and severity of follicular cell hyperthrophy in all animals at concentrations higher than 500 ppm. Histopathological lesions in the thyroid gland were associated with a disruption of thyroid hormone biosynthesis as shown by the increase in TSH levels in males and females and a concomitant decrease in T4 levels in males at 500 and 2500 ppm.

The absolute and relative weights of adrenal glands were statistically significantly increased in males at 500 and 2500 ppm, and females at 100 and 2500 ppm. At the microscopic level, changes were reflected by an increased diffuse vacuolation in the zona fasciculata and glomerulosa of the adrenal cortex. Similar changes were noted in animals at 500 ppm, affecting both the zona fasciculata and glomerulosa in females but only the zona fasciculata in males.

Macroscopic evaluation of the kidney in animals treated at 2500 ppm revealed a dark colour, which was closely related to the golden brown droplets found in the cytoplasm of the renal tubular epithelial cells in animals of this group.

The unscheduled, treatment-related deaths in males of the 2500 ppm group showed enlarged livers with dark appearance and white foci on the lobes. Other macroscopic findings included the presence of dark content in the intestine and haemorrhage around the genital area. At microscopic examination of the livers, acute focal or multifocal centrilobular to midzonal hepatocellular necrosis associated with mixed cell filtrate was observed. Consistent with the findings in all surviving animals of this dose group, diffuse centrilobular to panlobular hepatocellular hypertrophy was noted.

Based on the study results, the NOAEL for rats was established at 100 ppm, corresponding to 9.2 and 9.6 mg/kg bw/day in males and females, respectively.

 

In a previous subacute toxicity study, possible hepatotoxic effects of the test substance were investigated in male and female C57 Bl/6N Crl BR mice similar to OECD guideline 407 and complying with GLP (M-171883-01-3, 1999). Animals (15/sex/group) were fed ad libitum with diets containing 50, 250, 1000 and 2500 ppm of test substance for at least 28 days, corresponding to 9.3, 47.4, 186.2 and 458.0 mg/kg bw/day in males and 11.8, 57.9, 234.4 and 513.0 mg/kg bw/day in females. One group, consisting of 15 males and 15 females, received plain diet and served as controls.

Twenty-seven mortalities occurred during the study period: 1 female of the control group, 1 male and 4 females at 1000 ppm and 9 males and 12 females at 2500 ppm. Among the animals which died in the 1000 ppm dose group, the death of the male and 3 females were considered to be due to the erroneous administration of 2500 ppm from Day 8 to Day 11. The death of the fourth female in the 1000 ppm group was due to an accidental trauma. Neurobehavioural effects occurred at 2500 ppm and involved an occasional increase in motor activity in the animals.

Selected clinical chemistry parameters indicative of hepatotoxicity were analysed in plasma. A dose-related decrease in total bilirubin was observed in males at ≥ 50 ppm and in females at ≥ 250 ppm. The activity of aspartate and alanine aminotransferase was markedly increased in males at 1000 and 2500 ppm, being indicative of hepatic lesions.

At study termination, statistically significant and dose-related increase in absolute and relative liver weights was noted at ≥ 250 ppm in both sexes.

Enlargement and pale appearance of the liver were noted in males treated at 1000 and 2500 ppm and in all females at 2500 ppm. Slight to mild hepatocellular hypertrophy was found in liver, affecting either all zones of the hepatic lobule or only the centrilobular (males) or periportal (females) zones of liver. Hepatic vacuolation was observed at 250 and 1000 ppm in both sexes. In some males of the 250 ppm group and almost all males treated at 1000 ppm, scattered small foci of hepatocellular necrosis were present.

Analysis of microsomes prepared from the liver showed that the test substance induced a dose-related and significant increase in total liver cytochrome P450 content in males at 1000 and 2500 ppm compared to controls

The NOAEL for mice derived from the results of this study was 50 ppm, corresponding to 9.3 and 11.8 mg/kg bw/day in males and females, respectively.

 

In a non-GLP subacute toxicity study comparable to OECD guideline 407, 2 male and 2 female dogs per sex and group were fed ad libitum with diets at concentrations of 70, 200 and 700 ppm for 28 days in order to find appropriate dose levels for subsequent toxicity studies (M-171416-02-1, 1998). The corresponding mean dose levels over the study period were 2.4, 5.3 and 9.4 mg/kg bw/day in males and 2.5, 6.4 and 13.2 mg/kg bw/day in females. A further group, consisting of 2 males and females each, were administered plain diet and served as controls.

All animals of the 700 ppm group were sacrificed before study termination, since they suffered from treatment with the test substance. This was reflected by a decrease in food consumption and concomitant loss in body weight in all animals sacrificed at Days 14 and 15. A decline in body weight was observed in animals at 200 ppm, but food consumption was unaffected at this concentration.One male treated at 700 ppm showed reduced motor activity, irritability, dysrhythmia, prostration, tremors and absence of turning reflex.

Based on the results of this study, the NOAEL for beagle dogs was found to be at 200 ppm, which corresponds to 5.3 and 6.4 mg/kg bw/day in males and females, respectively.

 

Subchronic

A subchronic oral toxicity study in Wistar rats was performed according to OECD guideline 408 and in conformity with GLP (M-192116-02-2, 2000). The test substance was fed ad libitum to groups of 10 male and 10 female rats at 5, 20, 500 and 2500 ppm in the diet for 13 weeks. The corresponding mean dose levels over the study period were 0.3, 1.2, 30.5 and 154.8 mg/kg bw/day in males and 0.4, 1.5, 37.6 and 187.9 mg/kg bw/day in females. A similarly constituted group of 10 males and 10 females received untreated diet and acted as a control.

Eleven treatment-related mortalities were observed during the study, which occurred at dietary concentrations of 500 (1 male and 1 female) and 2500 ppm (8 males). A general pallor before death was noted in males which died at 2500 ppm. No treatment-related clinical signs were observed, except for the occasional incidence of piloerection, increased and/or reduced motor activity and irritability to touch. At 2500 ppm, body weight gains were reduced compared to controls during Week 1-5 in males and Week 1 in females, which corresponded to the decreased food consumption in males during the first 3 weeks and in females during the first week at this dose level.

Haematological analysis revealed decreased red blood cell parameters (e.g. haemoglobin, haematocrit) and increased prothrombin times at 500 and 2500 ppm in both sexes. Changes in clinical chemistry parameters mainly involved an increase in cholesterol at concentrations ≥ 20 ppm in females and at 2500 ppm in males. Furthermore, total protein and calcium concentrations were increased at ≥ 500 ppm in both sexes. Minor changes included the disturbance of mineral balances in animals at 2500 ppm (increased potassium) and females at 500 and 2500 ppm (decreased chloride).

Analysis of thyroid hormones in blood at Weeks 2, 6 and 13 revealed a statistically significant increase in TSH levels (thyroid stimulating hormone) in animals at ≥ 500 ppm, which was accompanied by a decrease in T4 (thyroxine) levels in males at this time points and concentrations. In females at ≥ 500 ppm, T4 levels were only statistically significantly decreased at Week 6. No treatment-related effects were seen in T3 (triiodothyronine) levels at Week 2 in both sexes. In the 2500 ppm group, a slight statistically significant increase in T3 levels was observed in females at Week 6 and 13 and in males at Week 13.

Organ weights of liver and thyroid gland were statistically significantly increased in males and females at dietary concentrations of 500 and 2500 ppm. All other changes in organ weights were considered to be incidental and occurred in the absence of corroborative histopathological changes.

Macroscopic changes consisting of enlargement and dark colouration of liver were observed in both sexes at 500 ppm and in a majority of animals at 2500 ppm. These findings correlated with the increased incidence and severity of centrilobular to generalized hepatocellular hypertrophy, which was more pronounced in females at microscopic evaluation. A golden brown pigment was noted in the cytoplasm of the sinusoidal lining cells or hepatocytes of most animals treated at 2500 ppm and some females at 500 ppm. Single cell necrosis was occasionally observed in animals of all test groups. Animals which died at 2500 ppm exhibited centrilobular hepatocellular necrosis. In kidneys, dark colouration was seen in animals at 2500 ppm, which was associated with an increased incidence of tubular golden brown pigment in the renal cortex. The thyroid gland was enlarged in some animals of the 500 and 2500 ppm group, which correlated with the increased thyroid weight and incidence of mild to marked follicular hypertrophy/hyperplasia in these animals. Although slight histopathological changes in thyroid also occurred in some animals treated at 5 and 20 ppm, these changes were not considered to be toxicologically significant, since no alterations in thyroid weights and hormone levels were seen.

Based on the effects observed during this study, the NOAEL for rats was considered to be 20 ppm, corresponding to 1.2 and 1.5 mg/kg bw/day for males and females, respectively.

 

In a previously conducted non-GLP subchronic toxicity study performed according to OECD guideline 408, Wistar rats in groups of 10/sex/dose were administered dietary levels of 10, 100, 500, 1000 and 5000 ppm for 13 weeks (M-210434-01-1, 1996b). The corresponding mean dose levels over the study period were 0.6, 6.3, 31.9 and 62.2 mg/kg bw/day in males and 0.8, 7.6, 37.6, 75.5 and 372 mg/kg bw/day in females. Due to mortality in the high dose in males (5000 ppm), no corresponding dose level could be calculated from the reported body weight and food intake values. A control group of 10 males and 10 females received plain diet.

At 5000 ppm all male animals died. In these animals, clinical signs involved a general pallor, piloerection, hunched posture, reduced motor activity, coldness to touch, prostration or dyspnoea. Body weight gain was statistically significantly decreased in females at 5000 ppm.

Haematology in females showed decreases in haemoglobin at ≥ 1000 ppm and in mean corpuscular volume at ≥ 500 ppm. Increased platelet counts were observed in females at ≥ 500 ppm and higher prothrombin time was noted in males at 500 and 1000 ppm. In clinical chemistry, bilirubin was decreased at ≥ 500 ppm in males and ≥ 100 ppm in females. Increases in cholesterol levels were seen at ≥ 500 ppm in females. Thyroid hormone analysis showed decreased levels of T4 associated with increased levels of TSH in animals treated at ≥ 500 ppm. Hepatocellular hypertrophy was observed at ≥ 100 ppm in males and ≥ 500 ppm in females associated with increased liver weights at ≥ 500 ppm in both sexes. Thyroid follicular hypertrophy/hyperplasia was observed at ≥ 100 ppm in both males and females.

Based on the effects observed in liver and thyroid gland, the NOAEL for rats was set at 10 ppm, corresponding to 0.6 and 0.8 mg/kg bw/day in males and females, respectively.

 

In a further subchronic non-GLP study according to OECD guideline 408, OF-1 mice (10/sex/group) were fed diets containing 10, 100, 500, 1000 and 5000 ppm of the test substance for 13 weeks (M-192384-01-1, 1996a). Corresponding mean dose levels over the study period were 1.5, 15.9, 79.4, 155.5 and 766.3 mg/kg bw/day in males and 2.0, 20.7, 103.1, 210.8 and 958.2 mg/kg bw/day in females. A control group (10 animals per sex) received plain diet.

Two treatment-related deaths occurred during the study at concentrations of 500 (1 male) and 5000 ppm (1 female). Clinical chemistry analysis revealed a decrease in bilirubin in males and an increase in cholesterol in females at 5000 ppm. Liver weights were significantly increased in males at ≥ 500 ppm, which correlated with centrilobular hypertrophy and microvacuolation of hepatocytes. At 5000 ppm, increased liver weights associated with diffuse hepatocellular hypertrophy were observed in females and centrilobular single cell necrosis and midzonal single cell fatty change were noted in males.

Based on the results of this study, a NOAEL of 100 ppm was derived for mice, corresponding to 15.9 and 20.7 mg/kg bw/day in males and females, respectively.

 

In beagle dogs, the subchronic toxicity of the test substance was investigated in a GLP-study following OECD guideline 409 (M-206098-02-2, 2001c). Groups of 4 animals per sex were administered concentrations of 30, 90 and 200 ppm via feed over a period of 13 weeks. The mean dose levels achieved during the study were 1.0, 3.2 and 7.6 mg/kg bw/day in males and 1.1, 3.6 and 8.5 mg/kg bw/day in females. An additional group of 4 animals per sex was fed plain diet and served as controls.

One female at 200 ppm was sacrificed due to treatment-related clinical signs including bloody faeces, diarrhoea, soiled fur, prostration, tremors and increased salvation. Lower body weight and body weight gains compared to controls were observed in males treated at 90 and 200 ppm. At Weeks 7 and 13, statistically significant increases in alkaline phosphatase activity were observed in females of the 200 ppm group. At microscopic examination, thymic athrophy was observed in males at ≥ 90 ppm and females at ≥ 200 ppm. In some males, slight to mild centrilobular hepatocellular hypertrophy was found at 90 and 200 ppm. Haemorrhages in stomach, gallbladder, duodenum, cerebellum and spinal cords were seen at microscopic evaluation of the female sacrificed.

Based on the overall effects of this study, the NOAEL for beagle dogs was considered to be 30 ppm in males and 90 ppm in females, which corresponded to doses of 1.0 and 3.6 mg/kg bw/day in males and females, respectively.

 

Chronic

The long-term effects of the test 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 chronic phase of the study, the dietary dose levels corresponded to a mean achieved compound intake of 0.25, 0.98, 3.73 and 12.39 mg/kg bw/day in males and 0.34, 1.34, 5.01 and 16.51 mg/kg bw/day in females, respectively (dose levels as calculated from the reported body weight and food intake values during Weeks 1-48). 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.

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 0.98 mg/kg/day in males and 1.34 mg/kg/day in females (20 ppm in the diet, Weeks 1-52).

 

In addition, the potential chronic toxicity of test substance was investigated in a GLP-conform study according to OECD guideline 452 and fulfilling the requirements of OECD guideline 409 (M-204326-01-2, 2001b). Five male and 5 female beagle dogs per test group received the test substance at 9, 30 and 90 ppm in the diet during a period of 52 weeks. The mean achieved dosages in this study were 0.27, 0.70 and 2.73 mg/kg bw/day in males and 0.22, 0.76 and 2.51 mg/kg bw/day in females. A control group consisting of 5 animals per sex was fed with plain diet.

No treatment-related mortalities and clinical signs were observed in the course of the study. At a dietary concentration of 90 ppm, a slight decrease in body weight gain was noted in both genders, but food consumption in this test group was not altered. Ophthalmological examination and urinalysis did not reveal any treatment-related changes in animals at all concentrations tested.

Changes in haematological parameters (e.g. higher thrombocyte level at Week 12 in males at 90 ppm, higher basophil level at Week 12 in females at 9 ppm, lower mean cell haemoglobin concentration in week 51 in females at 30 and 90 ppm, decreased APTT values) were considered to be unrelated to test substance treatment, since they were only slight or not always dose-related, and remained within the range of the historical background data. Similarly, alterations in clinical chemistry parameters (chloride levels, ASAT activity, cholesterol levels, ALP activity, ALAT activity, creatinine activity and amylase activity) only occurred occasionally and without clear dose-relationship. Therefore, these alterations were not considered to be of toxicological significance.

Organ weights in the treated animals did not differ from those of the controls. At necropsy, no treatment-related findings were observed after macroscopic and microscopic examination of treated animals compared to controls.

Based on the overall results of this study, the NOEL for beagle dogs was established at a concentration of 30 ppm, corresponding to 0.70 and 0.76 mg/kg bw/day in males and females, respectively.

 

Dermal

Subacute

The subacute dermal toxicity of the test substance was investigated in male and female albino (outbred) VAF/Plus® CD® (Sprague-Dawley derived) Crl:CD® (SD)IGS BR rats according to OECD guideline 410 and under conditions of GLP (M-210382-01-1, 2002). In this study, the test substance was dermally applied for 7 days/week during a period of 4 weeks to groups of 10 animals (5 males and 5 females) at dose levels of 10, 50, 250 and 1000 mg/kg bw/day (nominal doses). On each treatment day, amounts of the test substance corresponding to the respective nominal doses were placed onto the animal skin using a moistened patch and a semi-occlusive bandage. A control group of 5 male and female rats was sham-exposed using a patch moistened with 1.0 mL saline, which was fixed with a semi-occlusive bandage. Animals were daily exposed for 6 h, then wrappings were removed and the test substance was removed by washing.

All animals survived during the 28-day study period. No clinical signs of toxicity were observed in all animals. No effects were noted at the neurobehavioural and ophthalmoscopic examinations. Body weights, food consumption and urinalysis parameters remained unchanged in all animals.

Test substance-related changes in haematology and coagulation parameters compared to controls were found at 50, 250 and 1000 mg/kg bw/day in both genders. At these dose levels, mean haemoglobin concentrations, haematocrit values and/or erythrocyte counts were statistically significantly decreased, whereas activated partial thromboplastin time was statistically significantly increased. Prothrombin times were prolonged in males at ≥ 250 mg/kg bw/day and in females only at the high dose tested. Platelet counts were generally increased in males and females at 50, 250 and 1000 mg/kg/day. However, only the increases in males at 50 mg/kg/day and in females at 250 and 1000 mg/kg/day were statistically significant. A few statistically significant decreases in the counts of leukocytes, absolute lymphocytes and eosinophils were noted at the high-dose group (1000 mg/kg/day) in females, but these slight changes were not considered to be biologically significant. At study termination, test substance-related changes in clinical chemistry parameters compared to controls were observed in males and females at 250 and 1000 mg/kg/day. These involved alterations in clinical parameters indicative of hepatotoxicity such as increased activity of the liver enzyme gamma-glutamyl transferase and cholesterol levels. The activity of the hepatic enzyme alanine aminotransferase was only significantly increased in males receiving the high dose. Furthermore, increases in the serum levels of total protein and globulin, and concomitant decreases in albumin/globulin ratios were observed for males and females at 250 and 1000 mg/kg bw/day. In males, glucose was significantly decreased at 1000 mg/kg bw/day. Further changes in clinical parameters involved a statistically significant reduction in the activity of alkaline phosphatase in females treated with ≥ 250 mg/kg/bw/day and males treated with 1000 mg/kg bw/day. The biological significance of this finding was not explainable, since only high alkaline phosphatase activity may be indicative of hepatic changes. However, lower alkaline phosphatase may also be attributable to pathophysiological states resulting from malnutrition and hypothyroidism.

Treatment-related changes were found in the absolute and/or relative organ weight of liver in males and females at dose levels of 50, 250 and 1000 mg/kg bw/day. In addition, absolute and relative organ weights of the adrenal glands were increased in females receiving the test substance at 250 and 1000 mg/kg bw/day. No changes in the weights of the other organs were observed at the end of the treatment period.

At necropsy, brown discolouration and scattered tan foci in livers of males and females at dose levels higher than 50 mg/kg bw/day were noted. These macroscopic changes in the liver were considered to be related to treatment. In accordance with the observed increase in hepatic organ weights, histopathological examination of the liver revealed a test substance-related increase in the incidence and severity of centrilobular hepatocellular hypertrophy in males and females at doses ≥ 50 mg/kg bw/day. At the same dose levels, an increased incidence and severity of follicular cell hypertrophy/hyperplasia was seen in the thyroid gland in both genders. Furthermore, a dose-related increase in the incidence and severity of hypertrophy and hyperplasia was found in the zona fasciculate of the adrenal cortex in females, which is consistent with the observed increase in organ weights of the adrenal glands at these doses.

Based on the overall results of the study, the NOAEL was considered to be 10 mg/kg bw/day in male and female rats.

 

 

 

 

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)

 

Justification for classification or non-classification

The available data on the repeated dose toxicity of the substance via the oral and dermal route do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.