Pyrasulfotole

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:

08 Feb - 12 Jul 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 1981

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-[2-methanesulfonyl-4-(trifluoromethyl)benzoyl]-1,3-dimethyl-1H-pyrazol-5-ol

EC Number:

609-256-3

Cas Number:

365400-11-9

Molecular formula:

C14H13F3N2O4S

IUPAC Name:

4-[2-methanesulfonyl-4-(trifluoromethyl)benzoyl]-1,3-dimethyl-1H-pyrazol-5-ol

Test material form:

solid: crystalline

Test animals

Species:

rat

Strain:

Wistar

Remarks:

HsdCpb:WU

Details on species / strain selection:

The rat is the recommended species for repeat-dose dermal toxicity studies. Animals of this strain have been used at the Bayer HealthCare AG for toxicological studies for many years. Historical data on their physiology and spontaneous alterations is available.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH Experimental Animal Breeders in Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males: 9 - 10 weeks, females: 13 - 14 weeks
- Weight at study initiation: males: 222 - 262 g (mean 244 g), females: 202 - 230 g (mean 213 g)
- Fasting period before study: no
- Housing: individually in Type IIIh cages on wood shavings
- Diet: PROVIMI KLIBA® 3883.0.15 (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least seven days

DETAILS OF FOOD AND WATER QUALITY: analysis was performed

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 50 - 60
- Air changes (per hr): more than 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 08 Feb 2005 To: 09 Mar 2005

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Remarks:

The test item was applied as undiluted solid onto a wet gauze pad.

Details on exposure:

TEST SITE
- Area of exposure: back and flank region, 6.0 x 5.0 cm = 30.0 cm²
- % coverage: greater than 10% of body surface area
- Type of wrap if used: the test item was applied to the moist gauze-layer of a "Cutiplast steril®" patch and secured in place with using Peha-Haft® placed on the rat's back; the gauze-layer was secured in place using "Peha-Haft®" cohesive stretch tape (approx. 8 x 23 cm).
- Time intervals for shavings: one day before the start of treatment and twice weekly afterwards

REMOVAL OF TEST SUBSTANCE
- Washing (if done): test item was removed with soap and water
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount(s) applied: 10, 100 and 1000 mg/kg bw/day;
males reveived 9.6 - 12.8, 7.3 - 10.8 and 15.5 - 19.8 mg/cm²/day; females received 8.0 - 8.4, 5.7 - 9.5 and 15.0 - 16.1 mg/cm²/day
- For solids, paste formed: no

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes ("Lomir Biomedical Inc." rat jacket)

Analytical verification of doses or concentrations:

no

Remarks:

(No dose formulation preparation or analysis was performed during the study, as the test item powder was applied to the rat skin without using any vehicle (only moistened with water)).

Details on analytical verification of doses or concentrations:

not applicable

Duration of treatment / exposure:

study period 28 days (males received 22 applications, females 23)

Frequency of treatment:

first three weeks: 5 days/week; thereafter: 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: Dose levels were selected and determined by the sponsor according to results obtained in previous studies.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Inspections on mortality and morbidity of the animals were done twice daily and on weekends / on public holidays once a day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A careful clinical examination including observation outside the home cage in a standard arena (OFO) was made once prior to treatment and daily (careful clinical examination) or weekly (OFO) thereafter up to necropsies in all animals.

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: The shaved skin areas were examined before starting the study and each day of treatment during the study.

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed before the study was started and at the beginning of each study week.

FOOD CONSUMPTION:
- Food consumption for each animal 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

WATER CONSUMPTION: Yes
- Time schedule for examinations: Individual water intake was determined once weekly from the start of the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examinations were performed before the start of the study and at the end of the treatment period.
- Dose groups that were examined: all (pre-treatment), groups 1 (control) and 4 (high dose) at the end of the treatment period

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood sampling was performed before the start of the study and towards the end of the treatment period (males day 25, females day 28).
- Anaesthetic used for blood collection: Yes (CO2/O2)
- Animals fasted: No
- How many animals: all
- Parameters examined: leucocyte count, differential blood count, erythrocytes, haemoglobin, haematocrit, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, mean corpuscular volume, thrombocytes, reticulocytes, hepatoquick

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood sampling was performed before the start of the study and towards the end of the treatment period (males day 25, females day 28).
- Animals fasted: No
- How many animals: all
- Parameters examined: alanine aminotransferse activity, albumin concentration, alkaline phosphatase activity, aspartate aminotransferase activity, total bilirubin concentration, cholesterol concentration, creatinine concentration, calcium concentration, chloride concentration, glucose concentration, potassium concentration, sodium concentration, inorganic phosphate concentration, total protein concentration, triglycerides, urea concentration

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals alive at scheduled necropsy as well as those killed in moribund state

HISTOPATHOLOGY: Yes
- adrenal glands, aorta, brain (cerebrum, cerebellum, brain stem), epididymides, esophagus, eyes, eyelids, exorbital lacrimal glands, femur, harderian glands, head: nasal cavity, heart, intestine (incl. Peyer's patches): duodenum, jejunum, iileum, caecum, colon, rectum, remaining intestine, kidneys, larynx, liver, lungs, lymph nodes: mandibular and mesenteric, optic nerves, ovaries, oviducts, physical identifier, pancreas, pharynx, pituitary gland, prostate, salivary glands (parotid, submandibular, sublingual), sciatic nerve, seminal vesicles (incl. coagulation glands), skeletal muscle (tigh), skin (mammary region), spinal cord (cervical, thoracic, lumbar), spleen, sternum, stomach (forestomach and glandular stomach), sestes, thymus, thyroid glands (with parathyroids), tongue, trachea, ureters, urethra, urinary bladder, uterus (with cervix), vagina, Zymbal's glands, organs and tissues with macroscopic findings, skin - treated, skin - untreated

ORGAN WEIGHTS: Yes
- adrenal glands, kidneys, liver, brain, testes, epididymides, heart, ovaries, spleen, thyroid gland (in fixed state), thymus, uterus

Statistics:

As basis for statistical evaluation of numerical data SAS 6.12 was used. The results for the groups that received the test compound were compared with those for the control group. The statistical evaluation of data related to laboratory investigations, body and organ weights as well as food and water intake is performed using SAS® routines.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Dermal irritation:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In mid-dose males a statistically significantly higher MCHC value was noted (320 g/L Ery vs. 314 in the control). In mid-dose females a statistically significantly higher MCH value was noted (17.4 pg vs. 16.9 in the control). Both differences were only mimimal in degree and showed no dependency. Therefore these changes were considered to be not treatment-related.

See Attachment 2 for summary data of haematological findings.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The activities of aspartate aminotransferase were decreased at 10 mg/kg bw/day and above and of alkaline phosphatase at 100 mg/kg bw/day and above in both sexes.
The concentrations of cholesterol, triglycerides, total protein and albumin were higher, and the concentrations of creatinine lower in males at 1000 mg/kg bw/day. In females, the concentration of bilirubin was higher at 100 and 1000 mg/kg bw/day.
The concentration of sodium was decreased at 100 mg/kg bw/day and above in both sexes.
Calcium was increased at 1000 mg/kg in females, and inorganic phosphate decreased in males at 10 mg/kg bw/day and above.
All these partly statistically significant differences were either of a minimal degree, and/or not dose-related, and/or were observed only in one sex, and/or were all in the range of historical values. Therefore, they were considered as not treatment-related.

See Attachment 3 for summary data of clinical chemistry findings and Attachment 5 for laboratory historical control data.

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

not examined

Description (incidence and severity):

Not applicable.

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable.

Immunological findings:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative liver weights were statistically significantly increased at 1000 mg/kg bw/day in both sexes.

Absolute mean liver weight was statistically significantly increased in females at 10 mg/kg bw/day. However, relative mean liver weight of these animals was similar to the control group. In the absence of liver weight effects at 100 mg/kg bw/day this finding was considered incidental in nature.

See Table 2 for mean liver weight data on Day 28. See Attachment 4 for summary data of organ weight findings.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Locally, no evidence of any findings was detected at the application site of the skin.
In the pancreas, focal degeneration was detected at 100 mg/kg bw/day and above in both sexes.
In the thyroid gland, follicular cell hypertrophy was detected in both sexes in all dose groups, including controls. However, an increased incidence occurred in males at 1000 mg/kg bw/day (5-5-4-9 affected animals with increasing doses). Furthermore, colloid alteration of the thyroid was detected with an increased incidence in males (1-4-6-10 affected animals with increasing doses). The incidence occurred together with an increasing grading in the upper dose groups.
Slight hepatocellular hypertrophy was observed at 1000 mg/kg bw/day, in males.
Hypertrophy of the pars distalis of pituitary was observed at 1000 mg/kg bw/day in males.

See Attachment 1 for summary data of histopathological (non-neoplastic) findings.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Details on results:

Thyroid findings (increased weight, histopathological changes comprising changes in colloid, follicular cell hypertrophy and pigment deposition in the follicular epithelium) are considered a non-adverse and rat specific phenomenon. No changes of the thyroid were noted in either mice or dogs, the other two species in which repeated-dose studies with histopathological examination of the thyroid were conducted with the test substance. As the test substance through inhibition of the HPPDase enzyme increases plasma tyrosine concentration in the rat, it is quite possible that some of this increased tyrosine is taken up by the thyroid and stored in the colloid, either as free tyrosine or through either increasing the synthesis of thyroglobulin or altering its composition in terms of number of tyrosine residues per thyroglobulin molecule.
In the absence of signs of altered thyroid functions (such as effects on body weights, fertility and gestation indices or effects on offspring performance in the developmental neurotoxicity study), the observed morphological changes are considered to be non-adverse.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Remarks:

equivalent to 15.0 mg/m2

Sex:

male/female

Basis for effect level:

other: No dermal irritation observed up to the highest dose tested

2

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects seen at 10 mg/kg bw/day

3

Key result

Dose descriptor:

LOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: histopathological effects in the pancreas at 100 mg/kg bw/day

Target system / organ toxicity

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day

System:

gastrointestinal tract

Organ:

pancreas

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

2

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

3

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

4

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day

System:

endocrine system

Organ:

pituitary gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Table 2: Mean liver weights on Day 28   Dose groups (mg kg bw/day)   control 10 100 1000 Males Absolute (g) 14.42 13.90 13.71 16.43* Relative to Body Weight (%) 4.23 4.48 4.33 4.98** Females Absolute (g) 8.49 9.20* 8.50 9.45** Relative to Body Weight (%) 3.75 3.94 3.75 4.19**

* = p<0.05, ** = p< 0.01 (Dunnett test)

Applicant's summary and conclusion

Conclusions:

The study was performed under GLP conditions and according to OECD TG 410 (adopted 1981).There were few effects of repeated dermal application of the test substance to either male or female rats. There were no local effects on either skinfold thickness or on signs of irritation. The local NOAEL was determined to be 1000 mg/kg bw/day (equivalent to 15 mg/m2 skin), while the systemic NOAEL was 10 mg/kg bw/day, based on histopathological effects observed at 100 and 1000 mg/kg bw/day in both males and females.

Based on the effects observed in the pancreas at 100 mg/kg bw/day, the substance is classified "STOT RE 2, H373, gastro-intestinal".