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

Repeated dose toxicity: dermal

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

Test guideline 2

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

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:

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, sham-exposed

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

1

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

2

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

Target system / organ toxicity

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.