4-allylveratrole

Endpoint summary

Description of key information

OECD 423 (Methyl Eugenol): LD50 (oral; rat) 2500 mg/kg bw

OECD 402 (Methyl Eugenol). LD50 (dermal; rat) > 2000 mg/kg bw

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

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

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

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).

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.

Preliminary study:

300 mg/kg body weight: There were no clinical sings and pre-terminal deaths.

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 500 mg/kg bw

Acute toxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

GLP compliance:

yes

Test type:

fixed dose procedure

Limit test:

yes

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

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system 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.

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.

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:

other: no clinical signs were observed

Gross pathology:

No gross pathological chenges were observed

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Additional information

ORAL TOXICITY

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.

DERMAL TOXICITY

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.

Justification for classification or non-classification

The results of the key studies (OECD423 and OECD402) indicate that there is no need to classify Methyl Eugenol for acute toxicity via oral and dermal routes according to CLP Regulation 1272/2008.