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Repeated dose toxicity: dermal

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Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Mar - 13 Aug 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:
adopted in 1981
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Thiram
EC Number:
205-286-2
EC Name:
Thiram
Cas Number:
137-26-8
Molecular formula:
C6H12N2S4
IUPAC Name:
tetramethylthiuram disulfide

Test animals

Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Froxfield Farms U.K. Ltd., Petersfield, Hampshire, England
- Age at study initiation: 11 - 13 weeks
- Weight at study initiation: group means were 2.599 - 2.736 kg (males) and 2.688 - 2.763 kg (females)
- Fasting period before study: no
- Housing: individually
- Diet: SQC rabbit diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16 - 21
- Humidity (%): 42 - 55
- Air changes (per hr): approx. 19
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 25 Mar 1992 To: 13 Aug 1992

Administration / exposure

Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: dorsal region, 12 x 8 cm
- % coverage: approx. 10% of the total body surface
- Type of wrap if used: impervious bandage consisting of gauze covered with "Elastoplast" elastic adhesive dressing backed with impervious "Sleek" plaster.
- Time intervals for shavings or clippings: 24 h before the first application of the test substance and clipping was repeated as necessary during the experiment period.

REMOVAL OF TEST SUBSTANCE
- Washing: warm water and then gently blotted dry
- Time after start of exposure: 6 h

TEST MATERIAL
- Constant dose level: yes (based on recent body weight)
- For solids, paste formed: no, but the test substance was moistened with distilled water (0.6, 1.8 and 6 mL for the dose groups of 100, 300 and 1000 mg/kg bw/day, respectively).

USE OF RESTRAINERS FOR PREVENTING INGESTION: no
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
6 h/day for 21 (males) or 22 (females) consecutive days
Frequency of treatment:
21 (males) or 22 (females) consecutive days
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Fasting period before blood sampling for clinical biochemistry: yes, overnight
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for mortality and morbidity on working days, once on weekends and public holidays, once daily for signs of ill health, behavioral changes or toxicosis.

DETAILED CLINICAL OBSERVATIONS: No

DERMAL IRRITATION: Yes
- Time schedule for examinations: immediately prior to the first daily application of the test substance and subsequently daily. The observations were scored according to a modified Draize scoring system.

BODY WEIGHT: Yes
- Time schedule for examinations: prior to dosing and subsequently at weekly intervals during the study.

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

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to termination (Week 3)
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes (overnight)
- How many animals: all animals of all dose groups
- Parameters checked: packed cell volume (PCV), haemoglobin concentration (Hb), erythrocyte count (RBC), total leucocyte count (WBC), differential leucocyte count (WBC, differentiating among neutrophils (N), lymphocytes (L), eosinophils (E), basophils (B) and monocytes (M)), platelet count, mean cell volume (MCV), mean cell haemoglobin concentration (MCHC), Thrombotest, cell morphology.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to termination (Week 3)
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes (overnight)
- How many animals: all animals of all dose groups
- Parameters checked: alkaline phosphatase activity (AP), alanine amino-transferase activity (ALT), aspartate amino-transferase activity (AST), urea concentration, creatinine concentration, glucose concentration, total bilirubin concentration, globulin concentration (Glob), albumin concentration (Alb), albumin/globulin ratio (A/G), total cholesterol concentration (Chol), total protein concentration, sodium (Na), potassium (K), chloride (Cl), calcium (Ca) concentrations, inorganic phosphorus concentration (P).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were sacrificed at study termination and a gross pathological examination was performed.

Organs weighed: adrenals, liver, kidneys, testes with epididymides/ovaries

HISTOPATHOLOGY: Yes
- Tissues examined: abnormal tissue, skin (treated and untreated), kidneys, liver
- Fixative: 10% buffered formalin
- Embedding media: Paraffin wax
- Section thickness: 4 µm
- Staining: haematoxylin and eosin
- Animals examined: All tissues of all animals in the control and high-dose group, the skin was examined in all treatment and control groups.
Statistics:
All analyses were carried out separately for males and females. The following tests were used for food and water consumption, bodyweight, relative organ weight and clinical pathology data: If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75%), the proportion of animals with values different from the mode was analysed by appropriate methods. Otherwise: Bartlett’s test was applied to test for heterogeneity of variance between treatments. Where significant (at the 1% level) heterogeneity was found, a logarithmic transformation was tried to see if a more stable variance structure could be obtained. If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out. If significant heterogeneity of variance was present, and could not be removed by a transformation, the Kruskal-Wallis analysis of ranks was used. Analyses of variance were followed by a Student’s ‘t’ test and Williams’ test for a dose-related response, although only the one thought most appropriate for the response pattern observed has been reported. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the ‘t’ test and Williams’ test (Shirleys’ test). Where appropriate for organ weight data, analysis of covariance was used in place of analysis of variance.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed.

Summarized results can be found in Attachment 1 in the attached background material.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
- 100 mg/kg bw/day: Weekly mean irritation indices of 0 (Week 1), 0.9 (Week 2) and 1.5 (Week 2) for males and 0 (Week 1), 0.5 (Week 2) and 1.0 (Week 2) for females.
- 300 mg/kg bw/day: Weekly mean irritation indices of <0.1 (Week 1), 1.2 (Week 2) and 2.1 (Week 2) for males and <0.1 (Week 1), 0.6 (Week 2) and 1.3 (Week 2) for females.
- 1000 mg/kg bw/day: Weekly mean irritation indices of <0.1 (Week 1), 1.5 (Week 2) and 2.6 (Week 2) for males and <0.1 (Week 1), 1.1 (Week 2) and 2.3 (Week 2) for females.

Slight or well-defined erythema with or without slight or well-defined oedema, were noted with greater frequency in the mid- and high-dose group. Reactions were observed initially in isolated incidences on Day 7 but over the following two weeks developed in the great majority of rabbits. These reactions were frequently accompanied by residual (brown) staining from the test substance and/or desquamation of the stratum corneum. The control group showed no signs of dermal irritation.

Summarized results can be found in Attachment 1 in the attached background material.
Mortality:
no mortality observed
Description (incidence):
There were no mortalities.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- 100 and 300 mg/kg bw/day: No effects on body weight were observed between the control and treatment groups.
- 1000 mg/kg bw/day: Statistically significantly reduced bodyweight gain was observed in females dosed compared to the control group throughout the treatment period. Cumulative body weight gain in females was 40.5% decreased compared to the control group.

Summarized results can be found in Attachment 1 in the attached background material.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- 100 and 300 mg/kg bw/day: No effects on food consumption were observed between the control and treatment groups.
- 1000 mg/kg bw/day: : Reduction in mean food consumption was measured for females throughout the study. These changes were statistically significant (p 0.05) in Week 1 and 2.

Summarized results can be found in Attachment 1 in the attached background material.
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were observed between the control and treatment groups. The only statistically significant differences were slightly increased RBC (females, at all dose levels, up to +14.9%, but not dose-related) and slightly increased MCHC (females, 1000 mg/kg bw/day, +3.1%). As these were minor changes and not dose related, they were not considered treatment related or adverse.

Summarized results can be found in Attachment 1 in the attached background material.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- 100 and 300 mg/kg bw/day: No effects on clinical chemistry were observed between the control and treatment groups.
- 1000 mg/kg bw/day: Liver enzymes AST (+361%), ALT (+356%) and cholesterol (+88%) levels were statistically significantly increased in females, AP was statistically significantly increased (males +59.4%, females +18%), all compared to the control group.

Summarized results can be found in Attachment 1 in the attached background material.
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 applicbale
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No dose-related effects were observed.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No dose-related effects were observed.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Minimal generalised/focal acanthosis of epidermis at the treated skin sites of all male and female rabbits were considered treatment-related. Other treatment-related changes were not observed between control and treatment groups.

Summarized results can be found in Attachment 1 in the attached background material.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed up to this dose level.
Key result
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
food consumption and compound intake
Key result
Dose descriptor:
NOAEL
Remarks:
dermal irritation
Effect level:
< 100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: dermal irritation was observed in all treatment groups.

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

The LOAEL per area was 2.7 cm². It was calculated using the lowest body weight (2.6 kg), the lowest dose group (100 mg/kg bw/day) and the exposure area of 12 x 8 cm.

Applicant's summary and conclusion

Conclusions:
The study was conducted according to OECD guideline 410 and under GLP. Under the conditions of the test, the test substance caused dermal irritation at all dose levels when applied dermally for three weeks to the intact skin of male and female rabbits (100, 300 and 1000 mg/kg bw/day). Treatment-related disturbances in body weight, food consumption and various biochemical parameters were observed for rabbits receiving the test substance at 1000 mg/kg bw/day. Thus, no NOAEL was set for local skin effects and the NOAEL for systemic effects was 300 mg/kg bw/day for males and females.