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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guideline
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:
Methomyl
EC Number:
240-815-0
EC Name:
Methomyl
Cas Number:
16752-77-5
Molecular formula:
C5H10N2O2S
IUPAC Name:
(E)-[1-(methylsulfanyl)ethylidene]amino N-methylcarbamate
Test material form:
solid: crystalline
Specific details on test material used for the study:
Methomyl technical
Lot #: DPX-X1179-512
Purity: 98.6%

Test animals

Species:
rabbit
Strain:
New Zealand White
Remarks:
HM:(NZW)fBR
Details on species / strain selection:
Rabbits are considered an acceptable species suited for conducting dermal absorption studies. Their use is designated as appropriate according to the testing guidelines for toxicological evaluation of pesticides by the dermal route of exposure. In addition, rabbits were used as the test species in the previous study, and use of the same species for this new study facilitated comparison of results. The New Zealand White strain was chosen because extensive background information is available from the literature, the supplier, and previous studies conducted at the test facility.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Hare Marland
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Approximately 10 weeks old
- Weight at study initiation: 1603.6-1973.9 g
- Housing: The rabbits were housed singly in suspended, stainless steel, wire-mesh cages
- Diet: Purina certified high fiber rabbit chow # 5325, approximately 125 g per day
- Water: ad libitum
- Acclimation period: Quarantined for 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 20±1°C
- Humidity: 40-60%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
other: deionized water
Details on exposure:
TEST SITE
- Area of exposure: Approximately 190 cm²
- % coverage: 10% of total body surface area
- Type of wrap if used: Wrapped with successive layers of porous dressing (stretch gauze and adhesive bandage)


REMOVAL OF TEST SUBSTANCE
- After an exposure period of approximately 6 hours, the bandages were removed and the test sites were washed with Ivory® soap and warm tap water, and the skin was patted dry.

TEST MATERIAL
- Amount applied: 1 mL
- Constant volume or concentration used: yes
- For solids, paste formed: yes

VEHICLE
- Amount applied: 1 mL

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
6 hours
Frequency of treatment:
daily for 21 days
Doses / concentrationsopen allclose all
Dose / conc.:
15 mg/kg bw/day
Dose / conc.:
30 mg/kg bw/day
Dose / conc.:
45 mg/kg bw/day
Dose / conc.:
90 mg/kg bw/day
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a previous 21-day dermal study conducted with the LOEL was reported to be 50 mg/kg and the NOEL to be 5 mg/kg. Based on the results of the previous study and the pilot study, the dosages for this 21-day dermal study were selected.
- Pilot study: A dermal pilot study was conducted with the test substance to compare the sensitivity of the assay presently used to determine cholinesterase activity to the assay used in the previous study and to determine the washing procedures to be used in the main study. Additionally, the results of pilot study were used to determine the highest dosage tested in the main study. Before exposure, approximately 0.5 mL of blood was collected from the jugular vein of 4 male rabbits. Baseline cholinesterase activity in plasma and red blood cells was assessed.
Each rabbit was treated once dermally at a dosage of 100, 250, or 500 mg/kg. The test site was covered with a porous wrap. A control animal was similarly treated with deionized water only. After an exposure period of approximately 6 hours, the bandages were removed. The test site of each rabbit was washed twice with warm water. The test site was wiped with a dry swab after each washing; the swabs were analyzed for the presence of test substance to determine if the method of washing removed all of the test substance. After washing, the skin was patted dry. After the exposure period, the rabbits were examined for clinical signs of toxicity and dermal response.
Approximately 1 hour after the end of the exposure period, approximately 0.5 mL of blood was collected from the jugular vein of the rabbits. The rabbits were then euthanized by injection with a barbiturate into the auricular blood vessel and exsanguinated. Brains were collected and weighed, frozen at approximately -70°C, and analyzed for brain cholinesterase activity. Cholinesterase was evaluated in plasma and red blood cells.
No clinical signs of toxicity or dermal irritation were observed. The rabbit dosed at 500 mg/kg exhibited inhibition in plasma (56% of control) and brain (61% of control) cholinesterase activity.
Analyses of the swabs indicated there was residual test substance on the skin of the rabbits after washing with water. Therefore, the rabbits were washed with Ivory soap and warm water during the main study.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Rabbits were checked daily for mortality and for signs of illness, injury, or abnormal behavior.

OBSERVATIONS: Before treatment and after test substance removal each day, the rabbits were observed for the following clinical signs of toxicity indicative of cholinergic effects: salivation, excessive urination, convulsions, tremors, muscle fasciculations, diarrhea, hyperactivity, and hyperreactivity.

DERMAL IRRITATION: The animals were also observed for dermal irritation before treatment and after unwrapping each day. The Draize Scale was used to score skin responses. Adjacent areas of untreated skin were used for comparison.

BODY WEIGHT: The rabbits were weighed 2 times each week (at approximately 3-4 day intervals) during the dosing period.

FOOD CONSUMPTION and EFFICIENCY: The amount of food consumed by each rabbit was determined weekly. From these determinations and body weight data, individual daily food consumption and mean food efficiency were calculated.

CLINICAL CHEMISTRY: One hour following the last exposure (day 21), approximately 0.5-1 mL blood was collected from the jugular vein of all study rabbits and evaluated for cholinesterase activity in plasma and red blood cells.
Sacrifice and pathology:
Following blood collection on day 21, all rabbits were euthanized by injection with a barbiturate into the auricular blood vessel and then exsanguinated. The rabbits were given a gross pathological examination. Brains were collected and weighed, frozen at approximately -70°C, and analyzed for brain cholinesterase activity. The remaining tissues were discarded without microscopic evaluation.
Statistics:
Body weights, body weight gains, and pathology data were analyzed by a one-way analysis of variance. Pairwise comparisons between test and control groups were made with Dunnett's test. Cholinesterase measurements were analyzed by the Jonckheere test for trend (p <0.05). Increases in the incidences Of clinical observations were evaluated by the Cochran-Armitage test for trend.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Pupillary constriction, cataracts were considered to be non test substance related and spontaenous. Mucoidal feces, blood in feces, diarrhea, injured leg were considered to be related to stress occured during dosing and washing procedures.
Other clinical signs including pallor of the eyes, raised area on the rib(s), conjunctivitis, appears not to be eating, shivering, and weakness were not considered toxicologically important. These signs were observed sporadically or with similar frequency in both controls and treated animals.
Dermal irritation:
effects observed, non-treatment-related
Description (incidence and severity):
Slight or mild erythema, purplish areas, epidermal scaling, eschar, scratches, cuts, nicks, raw areas, bruises, tape burn, scars, sloughing, and scabs were observed sporadically in male and female rabbits at all dosages. These observations, although suggestive of skin trauma, exhibited no dose-response relationship and/or were attributed to mechanical irritation resulting from the daily bandaging, unwrapping, and washing regimen.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant differences in mean body weights or mean body weight gains occurred during the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No significant differences in food consumption occurred during the study.
Food efficiency:
no effects observed
Description (incidence and severity):
No significant differences in food efficiency occurred during the study.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Decreases in cholinesterase activity measured in brain, erythrocytes, or plasma attributable to the test substance were not demonstrated under the conditions of this study. There were some statistically significant differences between treated and control male and female groups for brain cholinesterase activity but in the absence of a meaningful dose response these were not considered to be biologically adverse.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related gross lesions were observed at necropsy. Discoloration noted in the eyes of a female rabbit in the 90 mg/kg/day treatment group was considered to be spontaneous since no other rabbits exhibited this lesion.

Effect levels

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
90 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Key result
Dose descriptor:
LOEL
Effect level:
> 90 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Dermal NOEL (Rabbit): 90 mg/kg (highest dose tested)
Executive summary:

The test substance was applied to the shaved, intact skin of male and female New Zealand White rabbits for 21 consecutive days to evaluate the toxicity of the test substance and to evaluate the potential for cholinesterase inhibition in the blood and brain following the guideline U.S. EPA OPP 82-2. The daily exposure period was approximately 6 hours. Four groups of 6 male and 6 female rabbits were treated dermally with 15, 30, 45, or 90 mg/kg of test substance per day. A vehicle control group of 6 male and 6 female rabbits was similarly treated with deionized water only. Body weights and food consumption were measured. Clinical observations and dermal effects were recorded before treatment and after test substance removal each day during the study. On test day 21, blood was collected from each rabbit approximately 1 hour after the exposure period to determine erythrocyte and plasma cholinesterase activity. Following blood collection, all rabbits were sacrificed, a gross necropsy was performed, and the brains were collected, weighed, and later analyzed to determine brain cholinesterase activity.

No mortalities occurred during the study. There were no significant differences in mean body weight, mean body weight gains, food consumption, or food efficiency. No clinical signs of toxicity attributable to dermal exposure to test substance occurred during the study.

No test substance-related gross lesions were observed in any dose group during necropsy. Decreases in cholinesterase activity measured in brain, erythrocytes, or plasma attributable to the test substance were not demonstrated under the conditions of this study. There were some statistically significant differences between treated and control male and female groups for brain cholinesterase activity but in the absence of a meaningful dose response these were not considered to be biologically adverse.

The no-observed-effect-level was determined to be 90 mg/kg, the highest dose tested, for both male and female rabbits.