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Diss Factsheets

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Sept 2017- 13 Oct 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
4-allylveratrole
EC Number:
202-223-0
EC Name:
4-allylveratrole
Cas Number:
93-15-2
Molecular formula:
C11H14O2
IUPAC Name:
1,2-dimethoxy-4-(prop-2-en-1-yl)benzene
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
Source: Geniron Biolabs Pvt. Ltd.
No.93, Solur, Anekal-Thally Road, Anekal
Bengaluru – 562106, India

No. of groups : Two treatment group (G1-FTS & STS and G2-FTS & STS)

No. of animals : 3 animals (females) / treatment step

Age at treatment : 8 to 10 Weeks

Body weight range at treatment : 174.8 to 203.9 g Note: At the time of selection of animals for each treatment step, the weight variation did not exceed ± 20 per cent of the mean body weight of any previously dosed animals.

Identification : By rat accession number. Identification of individual rats was by cage card and turmeric colour body markings. The rat accession number was allotted during the course of the study. The temporary body
marking during acclimatization period was done with crystal violet.

Acclimatization : After physical examination for good health and suitability for experiment, the animals were acclimatized six days for G1-FTS, eight days for G1-STS, ten days for G2-FTS and fourteen days for
G2-STS before treatment. Animals were observed once daily during acclimatization period.

Females were nulliparous and non-pregnant.

Rats were housed under standard laboratory conditions, air conditioned with adequate fresh air supply (13.6 to 13.8 air changes/hour). Environment: with temperature 20 to 24°C, relative humidity 64 to 67%,
with 12 hours light and 12 hours dark cycle. The maximum and minimum temperature and relative humidity in the experimental room were recorded once daily. The relative humidity in the experimental room was calculated from dry and wet bulb temperature recordings.

Housing Rats were housed individually in standard polysulfone cages (Size: approximately L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate
bottle. Additionally, polycarbonate rat huts were placed inside the cage as an enrichment object and were changed along with the cage once a week. Bedding: steam sterilized corn cob was used and changed once a week along with the cage.

Diet: ad libitum Hypro rat & mice pellet feed, manufactured by Krishna Valley Agro Tech LLP, MIDC Kupwad block, Sangli, Maharashtra, was provided to animals.

Water: ad libitum
Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd, Mumbai 400 001, India, was provided to animals in polycarbonate bottles with stainless steel sipper tubes. Water analysis report is enclosed as Annexure 3.

Animal selection
The body weight of all the animals was measured on the last day of acclimatization for the first treatment step and subsequently based on the body weight of first treatment step (G1-FTS), the rats with close body
weight range was randomly weighed and selected on the last day of acclimatization for subsequent next step of treatment group (G1-STS, G2-FTS and STS).


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Starting dose As per the public available data (PubChem), the acute oral LD50 of Methyl Eugenol for rats is 1179 mg/kg. Hence the test was started as per Annex 2c of the OECD 423 test guideline (Refer Annexure 1 of this report). The starting dose was 300 mg/kg body weight (G1 FTS).

As there was no test item-related mortality observed at the starting dose of 300 mg/kg body weight (G1 FTS); Hence test was continued with same dose of 300 mg/kg body weight with three additional female rats as second step (G1-STS). As there was no test item-related mortality in G1-STS, three additional animals were tested with 2000 mg/kg (G2-FTS) body weight as per Annex 2c of the OECD 423 test guideline. As there was no test itemrelated mortality in G2-FTS, three additional animals were tested with 2000 mg/kg (G2-STS) body weight as per Annex 2c of the OECD 423 test guideline. 1/3 rat was dead in G2-STS and based on the scheme - Annex 2c of the guideline OECD 423, the dosing was stopped. The subsequent dosing was done at approximately 48 to 94 hours after the previous dosing.
Doses:
300 mg/kg bw and 2000 mg/kg bw
No. of animals per sex per dose:
6 x 300 mg/kg bw (First and second treatment groups, 3+3), 6 x 2000 mg/kg bw (First and second treatment groups, 3+3 ). All animals were females
Control animals:
no
Details on study design:
Clinical signs and pre-terminal deaths

At each step, the animals were observed five times on test day 1 (day of administration) i.e. at 30 minutes and four times at hourly intervals and once daily during days 2 to 15 post administration. Observations included changes in skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern. Attention was directed to the observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma and all observed clinical signs were recorded.

Body weights
The body weights were recorded on test day 1 (pre-administration), day 8 (7 days post administration) and day 15 (14 days post administration).

Necropsy
The rats surviving to the end of the observation period were euthanised by using isoflurane anaesthesia and subjected to detailed necropsy. Gross pathological findings were recorded and reported. Microscopic examination was not carried out as no gross pathological changes were observed.

Results and discussion

Preliminary study:
300 mg/kg body weight: There were no clinical sings and pre-terminal deaths.
Effect levels
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
2 500 mg/kg bw
Based on:
test mat.
Mortality:
1 out of 6 rats in 2000 mg/kg bw dose group.
Clinical signs:
other: At 300 mg/kg no clinical signs observed. In 2000 mg/kg bw group: Hypoacticity and ataxia was observed during 30 minutes to 4 hours post dose on day 1. Two rats had slight/moderate dehydration on day 3. One preterminal death in 2000 mg/kg bw group.
Gross pathology:
No gross pathological changes detected at necropsy.

Applicant's summary and conclusion

Interpretation of results:
Category 5 based on GHS criteria
Conclusions:
The test item does not meet the criteria for classification as “Category 4” (300 mg/kg < Acute Toxicity Estimates ≤ 2000 mg/kg) as per Regulation (EC) No 1272/2008 of the European Parliament and of the council of 16 December 2008 on classification, labeling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006, as the LD50 cut off value is 2500 mg/kg body weight.

The test item is classified as “Category 5” (the oral LD50 range of 2000 to 5000 mg/kg) as per Globally Harmonized System of Classification and Labelling of Chemicals (GHS) Sixth Revised Edition, United Nations (2015). ST/SG/AC.10/30/Rev.6, as the LD50 cut off value is 2500 mg/kg body weight.
Executive summary:

The acute oral toxicity study with 4 -allylveratrole/Methyl Eugenol in Wistar rats was conducted to assess the toxicological profile of the test item.

Undiluted test item as supplied by the sponsor was administered at the dose of 300 mg/kg body weight (G1-FTS)] as a single oral gavage to overnight toxicity and pre-terminal deaths. Based on the scheme -guideline OECD 423, three additional female rats were tested at the same dose of 300 mg/kg body weight (G1-STS). There were no clinical signs of toxicity and pre-terminal deaths. Based on the scheme of the guideline OECD 423, three additional female rats were tested at next higher dose level of 2000 mg/kg body weight (G2-FTS). The clinical signs of hypoactivity, ataxia,slight/moderate/severe dehydration and posture (sitting with head hung down) were observed and all rats were normal from day 4 onwards and pre-terminal deaths. Based on the scheme of the

guideline OECD 423, the dose continued with the same dose of 2000 mg/kg body weight (G2-STS). The clinical signs of hypoactivity, ataxia and posture (sitting with head hung down) were observed and one rat died on day 2. The test item-related mortality was observed, and as per scheme no further test is required. Hence testing was stopped and the LD50 value was arrived.

The rats were observed for mortality and clinical signs for 14 days post treatment. Body weights were recorded prior to dosing on day 1 and again on days 8 and 15. Necropsy was performed for all the rats at termination.All survived rats gained weight during experimental period and decrease in body weight of dead rat. There were no gross pathological changes at necropsy.Based on the results of the present study, The LD50 cut off value is 2500 mg/kg body weight for the test item 4 -allylveratrole/Methyl Eugenol. The test item, Methyl Eugenol is classified as follows: · The test item is classified “Category 5” as per Globally Harmonized Classification system of Annex 2c of the Guideline, OECD 423.

No classification according to CLP regulation is warranted.