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

Repeated dose toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

446-630-3

EC Name:

-

Cas Number:

181587-01-9

Molecular formula:

C13H9Cl2F3N4OS

IUPAC Name:

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

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

20 ppm

Remarks:

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

Doses / concentrations 2

Dose / conc.:

100 ppm

Remarks:

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

Doses / concentrations 3

Dose / conc.:

500 ppm

Remarks:

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

Doses / concentrations 4

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.

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

1

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

2

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

Target system / organ toxicity

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.