Thiophanate-methyl

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:

May 8, 1990 - November 15, 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Analytical data on the stability of the test substance were not reported

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Thiophanate-methyl

EC Number:

245-740-7

EC Name:

Thiophanate-methyl

Cas Number:

23564-05-8

Molecular formula:

C12H14N4O4S2

IUPAC Name:

methyl N-{[2-({[(methoxycarbonyl)amino]methanethioyl}amino)phenyl]carbamothioyl}carbamate

Test material form:

solid: particulate/powder

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on species / strain selection:

The animal model, the New Zealand White rabbit, is recognized as appropriate for dermal toxicity studies and is a widely used strain for which significant historical control data are available.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazleton Research Products, Inc., Denver, PA.
- Age at study initiation: 15 weeks
- Weight at study initiation: 2.1 to 2.5 kilograms for the males and 2.1 to 2.3 kilograms for the females
- Fasting period before study: no
- Housing: all animals were housed individually in clean, wire-mesh cages
- Diet: ad libitum, Purina# Certified Rabbit Chow #5322
- Water: ad libitum, tap water
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY: Contaminants present in animal feed or water were not expected to interfere with the objectives of this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 67 ± 4
- Humidity (%): 58 to 78
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Type of coverage:

occlusive

Vehicle:

water

Details on exposure:

TEST SITE
- Area of exposure: The test material was applied to the shaved intact dorsal skin of each test animal.
- % coverage:
The test material covered approximately 2-3% of the total body surface on the animals in the 100 mg/kg/day group, approximately 5-6% of the total body surface on animals in the 300 mg/kg/day group and approximately 8-10% of the total body surface on animals in the 1000 mg/kg/day group.
- Type of wrap: Doses were applied under gauze binders and secured with nonirritating tape.
- Time intervals for shavings or clipplings: The animals were shaved twice weekly during the study to facilitate dosing and dermal observation.

REMOVAL OF TEST SUBSTANCE
- Washing: At the end of a six-hour exposure period, the dressings were removed and the test sites were washed with disposable paper towels moistened with deionized water.

TEST MATERIAL
- Amount(s) applied: Individual animal doses for the treated groups were moistened with deionized water to form a paste immediately prior to application. Immediately prior to application the individual doses were moistened with approximately 0.2, 0.5 and 1.5 milliliters of deionized water, respectively for the 100, 300 and 1000 mg/kg/day doses.
- Constant volume or concentration used: yes
- For solids, paste formed: yes

VEHICLE
- Amount(s) applied: The control material, deionized water, was administered at a dose volume of 1.0 mL/kg/day.

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

three weeks

Frequency of treatment:

six hours per day, five days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: for mortality and overt signs of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

DERMAL IRRITATION: Yes
- Time schedule for examinations: Each application site was examined for erythema, edema and other dermal findings once daily (immediately prior to dosing on days of dose administration). The sites were graded approximately 18 hours after removal of the wrappings. Erythema and edema were evaluated based on a four-step grading system of very slight, slight, moderate and severe. Other dermal findings, if present, were noted.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were measured weekly beginning one week prior to dose administration.

FOOD CONSUMPTION: Yes
- 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: day prior to study termination,
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: all
- Parameters checked: Total Leukocyte Count (White Cell), Erythrocyte Count (Red Cells), Hemoglobin, Hematocrit, Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Platelet Count, Differential WBC Count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day prior to study termination,
- Animals fasted: Yes
- How many animals: all
- Parameters checked: Glucose, Blood Urea Nitrogen, Blood Creatinine, Total Protein, Albumin, Albumin/Globulin Ratio (A/G Ratio), Sodium, Potassium, Chloride, Calcium, Phosphorus, Aspartate Aminotransferase (Aspartat Transfer), Alanine Aminotransferase (Alanine Transfer), Serum Alkaline Phosphatase (Alkaline Phoshatase), Total Bilirubin (Total Bili), Total Cholesterol, Globulin, Gamma Glutamyltransferase (Glutamyl Transfer)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Necropsy included examination of the external surface, all orifices, and the cranial, thoracic, abdominal and pelvic cavities including viscera. At the time of necropsy, the following tissues and organs were collected and preserved in 10% neutral buffered formalin: Kidneys (2), Liver (sections of two lobes), Skin (treated and untreated), Gross lesions

ORGAN WEIGHTS
The following organs were weighed from all animals at the scheduled sacrifice: brain, kidneys, liver, ovaries, testes
Paired organs were weighed together. Organ to final body weight ratios were calculated.

HISTOPATHOLOGY: Yes
Tissues listed were examined from animals in the control and high dose groups. In addition, treated skin, untreated skin, and gross lesions were examined from animals in the low and mid dose group.

Statistics:

All analyses were conducted using two-tailed tests for significance levels of 5% and 1% comparing the treatment groups to the control group by sex. All means were presented with standard deviations (S.D.) and the numbers of sampling units (N) used to calculate the means. All statistical tests were performed by a DEC (Digital Equipment Corporation) computer with appropriate programming. Analysis of body weights, body weight changes, food consumption, clinical pathology parameters and absolute and relative organ weight values were analyzed by a one-way analysis of variance, following by Dunnett´s test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical findings were limited. Decreased defecation was noted in two 1000 mg/kg/day group males and one 1000 mg/kg/day group female on the third day of dose administration and one other study day for each animal. The two 1000 mg/kg/day group males also had single occurrences of decreased urination noted. Head tilt was observed in one 100 mg/kg/day group male on the last 15 days of the study. This finding was not related to the test material.

Dermal irritation:

effects observed, non-treatment-related

Description (incidence and severity):

No dermal irritation was present in the control group. Signs of dermal irritation were limited in each treated group to sporadic occurrences of desquamation and/or very slight erythema, generally observed during the second week of test material administration. No dose-related patterns were apparent. No signs of edema were present in any dose group.
At a dose level of 100 mg/kg/day, two males had findings of very slight erythema on one or three days. Desquamation was noted for one 100 mg/kg/day male on one occasion. Two 100 mg/kg/day females had desquamation observed on one or three days. A single incidence of very slight erythema was noted for one 300 mg/kg/day male. Desquamation was present on a few days for three males and all females in the 300 mg/kg/day group. At 1000 mg/kg/day, a single occurrence of very slight erythema was noted for one male. All 1000 mg/kg/day group males had desquamation present on one to four occasions. Very slight erythema was noted on the last three study days for a single 1000 mg/kg/day female. Desquamation was noted on two females in the 1000 mg/kg/day group on a few days.
Males displayed no dermal effects after day 14. Except for 1000 mg/kg/day female no. 10986, females displayed no dermal effects after day 10.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test material-related effects on mean body weights or mean body weight gains. Mean body weights were slightly decreased in the 1000 mg/kg/day group males at weeks 1, 2 and 3 and mean body weight gains in these males appeared to be reduced beginning weeks 0 to 1. It should be noted that the comprehensive (weeks 0-3) mean body weight gain value was also reduced, however, this value was influenced by one male (no. 10957) with consistently lower body weight gains. When the comprehensive (weeks 0-3) body weight gain for male no. 10957 was excluded from the mean for the 1000 mg/kg/day male group, no remarkable differences were observed between the control group and the 1000 mg/kg/day male group. Therefore, the decreases in body weights and body weight gains were considered to be the result of biological variability (especially notable in small sample sizes). There were no numerical trends in mean body weights or body weight gains in the treated female groups when compared to the control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption (g/animal/day and g/kg bw/day) was significantly decreased at 1000 mg/kg bw/day throughout the study (20-30% compared to control). This was considered to be a possible treatment-related effect. It was however not of sufficient magnitude to produce a significant decrease in weight gain.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test material-related effects were seen in the hematology parameters at any dose level. All mean hematology values in the treated groups were comparable to the corresponding control group values. An evaluation of the mean absolute and relative numbers of leukocytes revealed statistically significant decreases (p<0.05 or p<0.01) in mean percentages and absolute numbers of monocytes in males dosed at levels of 100 and 1000 mg/kg/day. These differences were minimal, no dose response was apparent and therefore, the decreases were not considered to be toxicologically significant. No statistically significant differences in the numbers or types of leukocytes were observed for the 100 and 1000 mg/kg/day females or the 300 mg/kg/day males and females.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects were observed in the serum chemistry parameters. Some differences occurred between the treated groups and the control group when the values were compared, however, no trends were apparent and the differences were not statistically significant.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effect was apparent in the organ weight values at any dose level. The mean brain weight was increased (statistically significant at p<0.05) in the 300 mg/kg/day males when compared to the control group. All other mean organ weight values and organ weight relative to final body weight values in the 300 mg/kg/day male group were comparable to the control group. All mean organ weight values and organ weight relative to final body weight values were comparable to the control group for the 100 and 1000 mg/kg/day groups and the 300 mg/kg/day group females.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related lesions were observed at necropsy. Accessory spleens were present in the control and all treated groups. One control group male and one 1000 mg/kg/day female had white areas on the liver. Ovarian cysts were present in one 1000 mg/kg/day female. Reddened renal cortico-medullary junctions were observed in one 1000 mg/kg/day female. One 300 mg/kg/day group male had white foci on the liver. Another 300 mg/kg/day group male had a depressed area on the kidney. One 100 mg/kg/day male had pitted kidneys. White foci on the kidneys were noted for a single control group female.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Hyperkeratosis of the treated skin was observed for one female in each of the 100, 300 and 1000 mg/kg bw/day groups. The 300 mg/kg bw/day female also had nonsuppurative subdermal inflammation. No internal histopathological changes related to treatment were observed at dose levels of 100, 300 and 1000 mg/kg/day. Kidney changes (infiltrate lymphocyte, tubular necrosis, interstitial fibrosis, cortical infarct) and liver changes (infiltrate lymphocyte, fatty change, hyperemia, suppurative inflammation, hepatocellular necrosis) were generally seen at a higher incidence in the control group when compared to the high dose group. Several histopathologic changes were observed in other tissues and organs; these were also considered to be normal background changes.

Histopathological findings: neoplastic:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

food consumption and compound intake

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The NOAEL is considered to be 1000 mg/kg bw/day under the conditions of this study.

Executive summary:

A study according to EPA 82-2 and similar to OECD TG 410 was conducted. New-Zealand White rabbits were treated with the substance at doses of 100, 300 and 1000 mg/kg bw/day. The test material was moistened with deionized water and applied five days per week for three weeks to the shaved intact dorsal skin of each rabbit for a total of 15 applications per animal. The test material was applied under gauze binders and secured with non-irritating tape for a period of six hours per day. Each group consisted of five males and five females. A concurrent control group received deionized water (1.0 mL/kg bw) on a comparable regimen. All animals wore plastic Elizabethan cellars during the daily exposure period. The animals were observed for signs of overt toxicology, dermal irritation and effects on body weight, food consumption, clinical pathology parameters and organ weights. Complete necropsy examinations were performed on all animals. A microscopic examination was conducted on selected guideline tissues.

Analytical data on the stability of the test substance were not reported. No compound related effects were observed. No compound related effects were observed on body weight. Mean food consumption (g/animal/day and g/kg bw/day) was significantly decreased at 1000 mg/kg bw/day throughout the study (20-30% compared to control). This was considered to be a possible treatment-related effect. It was however not of sufficient magnitude to produce a significant decrease in weight gain. No compound related effects were observed. No compound related effects were observed on organ weights or during the gross pathology. Dermal irritation was limited to sporadic occurrence of very slight erythema and desquamation in all treated groups, generally during the second week of test material administration. Edema was not present in any treated group. Microscopic tissue changes were limited to the treated skin. Hyperkeratosis of the treated skin was observed in one female in each of the treated groups. Non-suppurative subdermal inflammation was also present for the 300 mg/kg bw/day female that had hyperkeratosis. Although there was an effect on food consumption that seems to be dose-related, it gave no significant effect on body weights or other investigated parameters and was therefore not adverse for the duration of the study. Therefore, the NOAEL(systemic and local) is considered to be 1000 mg/kg bw/day under the conditions of this study.