Registration Dossier

Administrative data

Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 April 2019 - 20 June 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
GLP compliance:
yes
Test type:
fixed dose procedure
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Name of Test Item : Methyl Eugenol
Chemical Name (IUPAC) : 4-allyl-1,2-dimethoxybenzene
CAS No. : 93-15-2
Physical Appearance
(with color) : Pale yellow liquid
Batch No. : 40003010419
Purity (As per Certificate of Analysis) : 99.24%
Date of Manufacture : Feb 2019
Date of Expiry : Jan 2021
Storage Condition : Ambient (21 to 29ºC)
Batch Produced by
(Name and address) : YASHO INDUSTRIES LIMITED
Plot No. 2514/2515, Phase IV, G. I. D. C.,
Vapi- 396195, Gujarat, India
Test Item Code by Test Facility : D819-001

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
Source of Supply : In-house bred animals
Body Weight Range at Receipt : Between 200 and 300 g
No. of Animals and Sex : Total of 05 females will be received (Females used will be nulliparous and non-pregnant)
Age at Treatment : 8 to 12 weeks
Animal Identification : Acclimatization period: All the animals will be identified by tail marking using a black permanent marker pen and cage cards.
Treatment period : The animals will be identified by writing last four digits of animal number on tail using a red permanent marker pen and cage cards.
Environmental Conditions : Animals will be housed under standard laboratory conditions, in an environmentally monitored air-conditioned room with adequate fresh air supply (12 to 15 air changes per hour), room temperature 22±3°C and relative humidity 30% to 70% with 12 hours fluorescent light and 12 hours dark cycle. The temperature and relative humidity will be recorded once daily.
Housing : Maximum three animals will be housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube. For range finding study animals will be housed individually after treatment. For main study, during treatment, the animals will be housed individually; and after patch removal, animals will be housed together. Clean sterilized paddy husk will be provided as bedding material. Paper shredding will be provided as enrichment.
Feed : Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) will be provided ad libitum to the animals throughout the experimental period. The contaminant analysis test report of the feed will be included in the study report.
Water : Water will be provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through Reverse osmosis unit will be provided in plastic water bottles with stainless steel sipper tubes.
Acclimatization : Healthy and young adult animals will be acclimatized for a minimum period of five days to laboratory conditions prior to dosing and will be observed for clinical signs once daily. Veterinary examination of all the animals will be performed on the day of receipt and on 5th day of acclimatization.

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
TEST SITE
- Area of exposure: not specified
- % coverage: 10
- Type of wrap if used: The area was covered with cotton gauze and held in place with non-irritating adhesive tape. The whole area was wrapped with a suitable semi-occlusive dressing (crepe bandage).

REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the contact period, the residual test item was washed using distilled water and dried with absorbent cotton.
- Time after start of exposure: 24h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The dose volume eas calculated based on the body weight
- Concentration (if solution): undiluted
- Constant volume or concentration used: no
Duration of exposure:
24h
Doses:
The dose levels of 200, 1000 and 2000 mg/kg body weight.
No. of animals per sex per dose:
Range finding Study:
200 mg/kg : 1F
1000 mg/kg : 1F
2000 mg/kg : 1F
Main study
2000 mg/kg : 2F
Control animals:
not required
Details on study design:
The study was performed in two phases that is range finding study and main study. Range finding study was performed with one animal and main study was performed with two animals.
The animals were dosed in a stepwise procedure with one female in range finding study. As the LD50 of the test item was not available a starting dose of 200 mg/kg body weight was selected from the fixed dose levels of 50, 200, 1000 and 2000 mg/kg body weight. Depending on the presence or absence of moribund conditions or mortality, further animals were treated as per the OECD 402 guideline Annex 2.
Dosing was sequential, allowing at least 48 hours between the testing of each step. The time interval between dosing at each level was determined by the onset, duration and severity of toxic signs.
Based on the outcome of range finding study, further dose was selected for the main study and treatment was carried out with two animals. Details of the step wise test procedure according to the OECD 402 guideline is presented as Annexure 1.
The test item was applied topically (dermal exposure). Required volume of the test item was calculated based on individual animal body weight and then undiluted test item was applied on the clipped area (approximately 10% of the total body surface area) and was covered with cotton gauze and held in place with non-irritating adhesive tape. The whole area was wrapped with a suitable semi-occlusive dressing (crepe bandage). The contact period of test item was 24 hours. At the end of the contact period, the residual test item was washed using distilled water and dried with absorbent cotton.
For example:
Volume applied (mL) = Dose (mg/kg)/1000 x Body weighyt (g) / Density (mg/mL)
All the animals were observed for clinical signs of toxicity and mortality at 20 to 30 min, 1 hr (±10 mins), 2 hrs (±10 mins), 4 hrs (±10 mins) and 6 hrs (±10 mins) post dosing on day 1 and thereafter at least once daily for clinical signs of toxicity and twice daily for mortality during the 14 days observation period. Individual animal body weight was recorded at receipt, on day 1 before test item application and on day 8 and 15 during the experimental period.
At the end of observation period on day 15, all surviving animals were humanely sacrificed by carbon dioxide asphyxiation and subjected to gross pathological examination.

Results and discussion

Effect levels
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
no indication of skin irritation up to the relevant limit dose level
Mortality:
no mortalility was observed
Clinical signs:
no clinical signs were observed
Body weight:
no changes in mean body weight and percent change in body weight were observed
Gross pathology:
No gross pathological chenges were observed

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions employed and based on the above results, it is concluded that the acute dermal median letha dose (LD50) of Methyl Eugenol in Sprague Dawley rats is > 2000 mg/kg bw.
Executive summary:

Methyl Eugenol was evaluated for acute dermal toxicity in Sprague Dawley rats as per the OECD Guideline 402. The study was performed in two phases i.e range finding study and main study. The study animals was dosed in a stepwise procedure with one female in range finding study. Range finding study was performed with three female rats (one rat per each dose) and main study was performed with two female rats. on the day before the application of the test item, fur on the dorso-lateral area of the trunk of the animals was removed by clipping closely with an electric hair clipper and care was taken to avoid abrading the skin. Required volume of the test item was calculated based on individual animal body weight and then undiluted test item was applied on the clipped area and was covered with cotton gauze and held in place with non-irritating adhesive tape. the whole are was wrapped with a sutable semi-occlusive dressing. The contact period of the test item was 24 hours. At the end of the contact period, the residual test item was washed using distilled water and dried with absorbent cotton.

No clinical signs and mortalities were observed at the dose level of 200 mg/kg bw. Further one animal was dosed at 1000 mg/kg bw. No clinical signs and mortalities were observed at the dose level of 1000 mg/kg bw in range finding study. Next one animal was dosed at 2000 mg/kg bw. No clinical signs and mortalities were observed at the dose level of 2000 mg/kg bw in range finding study. hence, during main study two animals were administered with the same dose level of 2000 mg/kg bw. No clinical signs and mortalities were observed at the dose level of 2000 mg/kg bw in the main study.

All animals were observed for clinical signs of toxicity and mortality at 20 to 30 min, 1 hrs, 2hrs, 4 hrs, and 6 hrs on treatment day 1 and thereafter once daily for clinical signs of toxicity and twic daily for mortality during the 14 days observation period. The body weight was recorded on day 1 before test item application and on day 8 and 15. At the end of observation period, all the animals were humanely sacrificed and subjected to necropsy and detailed gross pathological examination.

No mortality, clinical signs and skin reactions were noted. No treatment related changes in body weights and percent change in body weight with respect to day 1 were noted. Normalincrease in body weights were noted during the observation period. No treatment related gross pathological changes were noted in any of the dosed animals during necropsy.