Registration Dossier

Administrative data

Description of key information

Acute toxicity: lowest oral: LD50 =  541.5 mg/kg bw (RA to Isoeugenol, not OECD not GLP, WoE, rel. 4) in mice;
Acute toxicity: dermal: LD50 = 1911.6 mg/kg bw (Similar to OECD 402, K, rel. 2) in rabbits;
Acute toxicity: inhalation: worst-case classification as Harmful if inhaled.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Referenceopen allclose all

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
28 May to 31 October 1980
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Pre-guideline and pre-GLP study. Only basic data given for this range-finding study and only two animals used per dose (one male and one female), but considered sufficiently reliable in a weight of evidence for the purpose of hazard assessment.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 401 (Acute Oral Toxicity)
Deviations:
yes
Remarks:
no. of animals were lesser than 5 per dose (1/sex/dose); no details on environmental conditions, only 7 days observation period followed
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Remarks:
pre-GLP
Test type:
standard acute method
Limit test:
no
Species:
mouse
Strain:
not specified
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: not reported
- Age at study initiation: 4-5 weeks
- Weight at study initiation: 17-21 g
- Fasting period before study: for four hours
- Housing: individual cages
- Diet (e.g. ad libitum): not reported
- Water (e.g. ad libitum): not reported
- Acclimation period: not reported

ENVIRONMENTAL CONDITIONS: not reported

IN-LIFE DATES: not reported
Route of administration:
oral: gavage
Vehicle:
other: Groundnut oil
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 10% v/v in Groundnut oil

DOSE VOLUME APPLIED: 0.1, 0.2, 0.5, 1.0 and 2.0 mL/kg bw
Doses:
0.1, 0.2, 0.5, 1.0 and 2.0 mL/kg bw
No. of animals per sex per dose:
1/sex/dose
Control animals:
no
Details on study design:
- Duration of observation period following administration: 7 days
- Necropsy of survivors performed: Yes; all survivors were killed by CO2 asphyxiation and examined post mortem after 7 days. All animals dying were autopsied.
Statistics:
None
Preliminary study:
Not applicable
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
0.5 mL/kg bw
Based on:
test mat.
Remarks on result:
other: Corresponding to 450 mg/kg bw
Mortality:
- Mortality was observed in 1/2, 2/2 and 2/2 animals at 0.5, 1 and 2 mL/kg bw, respectively.
- No mortality was observed at 0.1 and 0.2 mL/kg bw.
Clinical signs:
Most mice were showing signs of stress within 30 minutes-1 hour after treatment. The mice dosed at 1 and 2 mL/kg bw, and one animal dosed at 0.5 mL/kg bw, became semicomatose/comatose, hypothermic and showed laboured breathing within 1 hour. All these animals died within 2-24 hours.
The surviving animal dosed at 0.1 and 0.2 mL/kg bw recovered within 18 hours.
Body weight:
Apart from the male mouse dosed at 0.5 mL/kg bw, all surviving animals gained weight during the 7 day observation period.
Gross pathology:
Autopsy of the mice that died revealed:
- Irritation of the stomach and small intestines, dark spleen, congested lungs and mottled liver.
- Histological examination did not reveal liver necrosis.
Other findings:
None

None

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
Under the test conditions, test material is classified Category 4 (H302: Harmful if swallowed) according to the Regulation (EC) No. 1272/2008 and of the GHS as the LD50 value is comprised between 300 and 2000 mg/kg bw.
Executive summary:

In an acute oral toxicity study, mice (1/sex/dose) were given a single oral (gavage) dose of test item at 0.1, 0.2, 0.5, 1.0 and 2.0 mL/kg bw. Animals were then observed for mortality, clinical signs and bodyweights for 7 days and were all sacrificed for macroscopic examination.

Mortality was observed in 1/2, 2/2 and 2/2 animals at 0.5, 1 and 2 mL/kg bw, respectively. No mortality was observed at 0.1 and 0.2 mL/kg bw. Most mice were showing signs of stress within 30 minutes-1 hour after treatment. The mice dosed at 1 and 2 mL/kg bw, and one animal dosed at 0.5 mL/kg bw, became semi-comatose/comatose, hypothermic and showed laboured breathing within 1 hour. All these animals died within 2-24 hours. The surviving animal dosed at 0.1 and 0.2 mL/kg bw recovered within 18 hours. Apart from the male mouse dosed at 0.5 mL/kg bw, all surviving animals gained weight during the 7 day observation period.

Autopsy of the mice that died revealed: Irritation of the stomach and small intestines, dark spleen, congested lungs and mottled liver. Histological examination did not reveal liver necrosis.

Mice oral LD50 = 0.5 mL/kg bw (corresponding to 541.5 mg/kg bw).

Under the test conditions, test material is classified Category 4 (H302: Harmful if swallowed) according to the Regulation (EC) No. 1272/2008 and of the GHS as the LD50 value is comprised between 300 and 2000 mg/kg bw..

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1964
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Pre-guideline and pre-GLP study. Only basic data given but considered sufficient for a weight of evidence (test system not reported; body weight and gross pathology not reported).
Principles of method if other than guideline:
Rats and Guinea-pigs were given a single oral dose of test item and then observed for 14 days.
GLP compliance:
no
Remarks:
pre-GLP
Test type:
standard acute method
Limit test:
no
Species:
other: Rats and Guinea-pigs
Strain:
other: Osborne-Mendel rats
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: rats: 180-350 g; guinea-pigs: no data
- Fasting period before study: 18 hours
- Diet, ad libitum
- Water, ad libitum
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Not reported
Doses:
No data
No. of animals per sex per dose:
rats: 5
guinea pigs: no data
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations: The rats were observed until the survivors had returned to normal in appearance and weight (no data for guinea pigs)
Statistics:
LD50s were computed by the method of Litchfield and Wilcoxon (1949).
Key result
Sex:
male/female
Dose descriptor:
other: Rat LD50
Effect level:
1 560 mg/kg bw
Based on:
test mat.
95% CL:
1 290 - 1 880
Key result
Sex:
male/female
Dose descriptor:
other: Guinea pig LD50
Effect level:
1 410 mg/kg bw
Based on:
test mat.
95% CL:
1 130 - 1 780
Mortality:
Rats: Animals died within 1 hour to 4 days after treatment.
Guinea-pigs: Animals died within 3 to 6 days after treatment
Clinical signs:
Rats: Comatose soon after treatment, scrawny appearance for several days.
Guinea-pigs: Depression and coma
Body weight:
No data
Gross pathology:
No data

None

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
Under the test conditions, test material is classified Category 4 (H302: Harmful if swallowed) according to the Regulation (EC) No. 1272/2008 and of the GHS as the LD50 value is comprised between 300 and 2000 mg/kg bw for rats and for guinea-pigs.
Executive summary:

In an acute oral toxicity study, rats (5/sex/dose) and guinea-pigs (number not reported) were administered the test substance by gavage with a stomach tube. Animals were then observed for mortality and clinical signs for 14 days.

In rats, coma was observed soon after treatment. Deaths occurred between 1 hour and 7 days and clinical signs were reported to be scrawny appearance and coma.

In guinea-pigs, deaths occurred between 3 and 6 days and clinical signs were reported to be depression and coma.

Rat oral LD50 = 1560 mg/kg bw (95% CL 1290-1880 mg/kg bw).

Guinea-pig oral LD50 = 1410 mg/kg bw (95% CL 1130 -1780 mg/kg bw).

 

Under the test conditions, test material is classified Category 4 (H302: Harmful if swallowed) according to the Regulation (EC) No. 1272/2008 and of the GHS as the LD50 value is comprised between 300 and 2000 mg/kg bw for rats and for guinea-pigs.

This study is considered as sufficiently reliable in a weight of evidence for the purpose o f acute oral toxicity endpoint.

Endpoint:
acute toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included in Iuclid Section 13.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar physico-chemical, toxicological and environmental fate properties because of their structural similarity (cis- and trans-isomers).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance is the trans isomer (E), as a mono-constituent substance, with cis isomer (Z) as an impurity. The source substance is a reaction mass, composed of two diastereoisomers (the source substance [trans] and its cis-isomer).

3. ANALOGUE APPROACH JUSTIFICATION
Source and target substances have a common major constituent (trans-isomer) and the impurity of the target substance is the second major constituent of the source substance.
The studies were performed before the adoption of OECD TGs. However, the study design is similar to the one of the OECD TG 401 based on the exposure conditions and the key parameters assessed so the results are considered adequate and reliable for the purpose of prediction. The test material was not clearly identified but it is considered to represent the source substance in terms of constituents and impurities. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the results of the acute oral toxicity studies conducted in both the rat and the mouse with the source substance are likely to predict the properties of the target substance and are considered as adequate to fulfil the information requirement of Annex VII, 8.5.1.
The study giving rise to the highest concern, i.e. the one giving the lowest value for the LD50 was used for the read-across. Therefore, there is no selection bias for study used for the prediction.

4. DATA MATRIX
Cf. Iuclid Section 13.
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Preliminary study:
Not applicable
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
0.5 mL/kg bw
Based on:
test mat.
Remarks on result:
other: Corresponding to 450 mg/kg bw
Mortality:
- Mortality was observed in 1/2, 2/2 and 2/2 animals at 0.5, 1 and 2 mL/kg bw, respectively.
- No mortality was observed at 0.1 and 0.2 mL/kg bw.
Clinical signs:
Most mice were showing signs of stress within 30 minutes-1 hour after treatment. The mice dosed at 1 and 2 mL/kg bw, and one animal dosed at 0.5 mL/kg bw, became semicomatose/comatose, hypothermic and showed laboured breathing within 1 hour. All these animals died within 2-24 hours.
The surviving animal dosed at 0.1 and 0.2 mL/kg bw recovered within 18 hours.
Body weight:
Apart from the male mouse dosed at 0.5 mL/kg bw, all surviving animals gained weight during the 7 day observation period.
Gross pathology:
Autopsy of the mice that died revealed:
- Irritation of the stomach and small intestines, dark spleen, congested lungs and mottled liver.
- Histological examination did not reveal liver necrosis.
Other findings:
None

None

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
Based on the available data on the analogue substance, the registered substance is classified Category 4 (H302: Harmful if swallowed) according to the Regulation (EC) No. 1272/2008 and of the GHS as the LD50 value is comprised between 300 and 2000 mg/kg bw.
Executive summary:

In an acute oral toxicity study, mice (1/sex/dose) were given a single oral (gavage) dose of test item at 0.1, 0.2, 0.5, 1.0 and 2.0 mL/kg bw. Animals were then observed for mortality, clinical signs and bodyweights for 7 days and were all sacrificed for macroscopic examination.

Mortality was observed in 1/2, 2/2 and 2/2 animals at 0.5, 1 and 2 mL/kg bw, respectively. No mortality was observed at 0.1 and 0.2 mL/kg bw. Most mice were showing signs of stress within 30 minutes-1 hour after treatment. The mice dosed at 1 and 2 mL/kg bw, and one animal dosed at 0.5 mL/kg bw, became semi-comatose/comatose, hypothermic and showed laboured breathing within 1 hour. All these animals died within 2-24 hours. The surviving animal dosed at 0.1 and 0.2 mL/kg bw recovered within 18 hours. Apart from the male mouse dosed at 0.5 mL/kg bw, all surviving animals gained weight during the 7 day observation period.

Autopsy of the mice that died revealed: Irritation of the stomach and small intestines, dark spleen, congested lungs and mottled liver. Histological examination did not reveal liver necrosis.

Mice oral LD50 = 0.5 mL/kg bw (corresponding to 541.5 mg/kg bw).

Under the test conditions, test material is classified Category 4 (H302: Harmful if swallowed) according to the Regulation (EC) No. 1272/2008 and of the GHS as the LD50 value is comprised between 300 and 2000 mg/kg bw..

Endpoint:
acute toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included in Iuclid Section 13.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar physico-chemical, toxicological and environmental fate properties because of their structural similarity (cis- and trans-isomers).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance is the trans isomer (E), as a mono-constituent substance, with cis isomer (Z) as an impurity. The source substance is a reaction mass, composed of two diastereoisomers (the source substance [trans] and its cis-isomer).

3. ANALOGUE APPROACH JUSTIFICATION
Source and target substances have a common major constituent (trans-isomer) and the impurity of the target substance is the second major constituent of the source substance.
The studies were performed before the adoption of OECD TGs. However, the study design is similar to the one of the OECD TG 401 based on the exposure conditions and the key parameters assessed so the results are considered adequate and reliable for the purpose of prediction. The test material was not clearly identified but it is considered to represent the source substance in terms of constituents and impurities. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the results of the acute oral toxicity studies conducted in both the rat and the mouse with the source substance are likely to predict the properties of the target substance and are considered as adequate to fulfil the information requirement of Annex VII, 8.5.1.
The study giving rise to the highest concern, i.e. the one giving the lowest value for the LD50 was used for the read-across. Therefore, there is no selection bias for study used for the prediction.

4. DATA MATRIX
Cf. Iuclid Section 13.
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Key result
Sex:
male/female
Dose descriptor:
other: Rat LD50
Effect level:
1 560 mg/kg bw
Based on:
test mat.
95% CL:
1 290 - 1 880
Key result
Sex:
male/female
Dose descriptor:
other: Guinea pig LD50
Effect level:
1 410 mg/kg bw
Based on:
test mat.
95% CL:
1 130 - 1 780
Mortality:
Rats: Animals died within 1 hour to 4 days after treatment.
Guinea-pigs: Animals died within 3 to 6 days after treatment
Clinical signs:
Rats: Comatose soon after treatment, scrawny appearance for several days.
Guinea-pigs: Depression and coma
Body weight:
No data
Gross pathology:
No data

None

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
Based on the available data on the analogue substance, the registered substance is classified Category 4 (H302: Harmful if swallowed) according to the Regulation (EC) No. 1272/2008 and of the GHS as the LD50 value is comprised between 300 and 2000 mg/kg bw for rats and for guinea-pigs.
Executive summary:

In an acute oral toxicity study, rats (5/sex/dose) and guinea-pigs (number not reported) were administered the test substance by gavage with a stomach tube. Animals were then observed for mortality and clinical signs for 14 days.

In rats, coma was observed soon after treatment. Deaths occurred between 1 hour and 7 days and clinical signs were reported to be scrawny appearance and coma.

In guinea-pigs, deaths occurred between 3 and 6 days and clinical signs were reported to be depression and coma.

Rat oral LD50 = 1560 mg/kg bw (95% CL 1290-1880 mg/kg bw).

Guinea-pig oral LD50 = 1410 mg/kg bw (95% CL 1130 -1780 mg/kg bw).

 

Under the test conditions, test material is classified Category 4 (H302: Harmful if swallowed) according to the Regulation (EC) No. 1272/2008 and of the GHS as the LD50 value is comprised between 300 and 2000 mg/kg bw for rats and for guinea-pigs.

This study is considered as sufficiently reliable in a weight of evidence for the purpose o f acute oral toxicity endpoint.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
541.5 mg/kg bw
Quality of whole database:
No study was identified on the registered substance (trans-Isoeugenol). However, two studies were available on the supporting substance (Isoeugenol, mixture of cis- and trans-Isoeugenol). Several species were tested. The two studies were not sufficiently reliable as such (Klimisch = 4) but were considered sufficiently reliable in a weight of evidence for the purpose of hazard assessment. The study having the lowest LD50 was taken as the key value for this endpoint.

Acute toxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
acute toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Reason / purpose:
data waiving: supporting information
Reason / purpose:
data waiving: supporting information
Reason / purpose:
data waiving: supporting information
Reason / purpose:
data waiving: supporting information
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Quality of whole database:
No standard acute inhalation toxicity study was available on the registered substance. However, relevant data from other inhalation studies were used to conclude on the acute hazard via inhalation.

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:
From November 21, 1978 to April 23, 1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
This study was performed prior to the OECD test guideline No. 402 but the protocol is similar to that guidance.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
yes
Remarks:
(occlusive dressing, 3 animals/sex used; no details on environmental condition of animal room)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Test type:
standard acute method
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: C.S.E. colony, Branchville, N.J.
- Weight at study initiation: 2-3.4 kg
- Housing: Animals were housed individually under standard laboratory conditions.
- Diet: Purina Rabbit Chow, ad libitum
- Water: Water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS: Animals were housed and maintained according to 'Guide for the Care and use of Laboratory Animals'. DHEW Publication No. (NIH) 78-23. Revised 1978.

IN-LIFE DATES: From: November 11 1978 To: March 1979
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
TEST SITE
- Area of exposure: Dorsal surface of the trunk
- % coverage: 20 % of the entire body surface
- Type of wrap if used: Opened plastic sleeves (Baggies) were placed over the trunk and the posterior end was taped against the animal. Test material was applied over the prepared skin and the anterior end of the sleeve was taped against the animal, allowing the central portion to balloon. Elastic tape (Elastikon) was then lightly wrapped around the sleeve to reduce the chance of puncture or dislocation of the sleeve.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Excess material was cleansed from the skin using a clean disposable napkin moistened with saline.
- Time after start of exposure: 24 h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.8, 1.25, 1.57, 1.98, 3.15 and 5 mL/kg bw
Duration of exposure:
24 h
Doses:
0.8, 1.25, 1.57, 1.98, 3.15 and 5 mL/kg bw
No. of animals per sex per dose:
3
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Animals were observed for signs of toxicity or mortality twice daily (once daily on Saturday and Sunday) for 14 days.
- Necropsy of survivors performed: yes; any dead animals and all survivors were subjected to gross necropsy.
Statistics:
The LD50, slope and fiducial limits of the combined male and female mortality were estimated by the graphic method of Litchfield and Wilcoxon (1949)
Preliminary study:
Not applicable
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
1.77 mL/kg bw
Based on:
test mat.
95% CL:
> 1.35 - < 2.32
Remarks on result:
other: Equivalent to 1911.6 mg/kg bw (fiducial limits 1458-2506 mg/kg bw)
Mortality:
- No mortality was observed at 0.8 mL/kg bw.
- 1/6, 2/6, 4/6, 6/6 and 6/6 animals died at 1.25, 1.57, 1.98, 3.15 and 5 mL/kg bw, respectively. The majority of the deaths occurred within 24 hours.
Clinical signs:
- Systemic toxicity occurred in animals at all doses except 0.8 mL/kg bw.
- At 1.25 mL/kg bw, skin became erythematous and haemorrhagic areas were developed. Following drying and thickening, the skin was healed by termination.
- At 1.57 mL/kg bw, intradermal haemorrhage and eschar formation, with recovery toward normal skin texture in survivors were observed. Weakness was noted in all animals. Laboured breathing was observed in the first week.
- At 1.98 mL/kg bw, breathing was rapid and noisy and some animals had nasal discharge after 24 h. Respiratory symptoms persisted to death, but there was slow resolution in survivors. Haemorrhage and eschar formation were observed at the site of application.
- At 3.15 mL/kg bw, no meaningful clinical signs were observed before death of animals.
- At 5 mL/kg bw, slight erythema over application site was developed within 3 h and the skin became dark and haemorrhagic in 24 h. Nasal discharge was observed in animals which died.
Body weight:
- All surviving animals showed normal bodyweight gain over the 14-day study period, except for three males (one each from 0.8, 1.25 and 1.98 mL/kg bw group) and one female treated with 0.8 mL/kg bw which showed body weight loss.
Gross pathology:
- At 5 mL/kg bw effects observed at necropsy were congestion of the lungs.
- At 3.15 mL/kg bw, the major effects observed at necropsy were congestion of the lungs and failure of the vascular system characterized by haemorrhage, either locally into treated skin or into visceral organs
- At 1.98 mL/kg bw the major effects observed at necropsy were eschar formation and bruising of the skin and congestion of the lungs.
- At 1.57 and 1.25 mL/kg bw, 2/6 and 1/6 rabbits had congested lungs, respectively.
- At 0.8 mL/kg bw, no meaningful lesions were detected at necropsy.
Other findings:
None

None

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
Under the test conditions, test material is classified Category 4 (H312: Harmful iin contact with skin) according to the Regulation (EC) No. 1272/2008 and to the GHS as the LD50 value is comprised between 300 and 2000 mg/kg bw.
Executive summary:

In an acute dermal toxicity study, groups of New Zealand White rabbits (3/sex/dose) were given a single dermal application of undiluted test material at 0.8, 1.25,1.57, 1.98, 3.15 and 5.0 mL/kg bw to intact skin on the back. Test sites were covered with an occlusive dressing for 24 h. Animals were observed for mortality, clinical signs and bodyweights for 14 days. Animals which died and all survivors were subjected to gross necropsy.

No mortality was observed at 0.8 mL/kg bw. 1/6, 2/6, 4/6, 6/6 and 6/6 animals died at 1.25, 1.57, 1.98, 3.15 and 5 mL/kg bw, respectively. Thickening of the skin and varying degrees of erythema were observed. Eschar formation developed and necrotic patches began to exfoliate at about Day 7, continuing until termination, and leaving a healthy healing skin under the slough. Systemic toxicity occurred in animals at all doses except 0.8 mL/kg bw. Animals had dyspnea (either slow and laboured or very rapid breathing), nasal discharge (ranging from blood-tinged and frothy to thick and opaque) and congested lungs were found upon necropsy. The incidence and severity of the signs were least in the lower dose groups. All surviving animals showed normal bodyweight gain over the 14-day study period, except for three males (one each from 0.8, 1.25 and 1.98 mL/kg bw group) and one female treated with 0.8 mL/kg bw which showed body weight loss.

Dermal LD50 Combined = 1.77 mL/kg bw (fiducial limits1.35-2.32 mL/kg bw) [equivalent to 1911.6 mg/kg bw (fiducial limits 1458-2505.6 mg/kg bw).

Under the test conditions, test material is classified Category 4 (H312: Harmful iin contact with skin) according to the Regulation (EC) No. 1272/2008 and to the GHS as the LD50 value is comprised between 300 and 2000 mg/kg bw.

This study is considered as acceptable and satisfies the requirement for acute dermal toxicity endpoint.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
1 911 mg/kg bw
Quality of whole database:
The key study performed in rats was pre-GLP, but was similar to OECD Test Guideline No 402. This study was considered sufficiently robust to cover this endpoint.

Additional information

Acute toxicity: oral

No study was identified on the registered substance (trans-Isoeugenol). However, two studies were available on the supporting substance (Isoeugenol, mixture of cis- and trans-Isoeugenol, see Iuclid section 13 for read-across justification). Several species were tested. Therefore, the studies were used in a weight-of evidence approach.

In the first study (URL, 1980, rel. 4), the mice LD50 was calculated to be 541.5 mg/kg bw (corresponding to 0.5 mL/kg bw). Mortality was observed in 1/2, 2/2 and 2/2 animals at 0.5, 1 and 2 mL/kg bw, respectively. No mortality was observed at 0.1 and 0.2 mL/kg bw. Most mice were showing signs of stress within 30 minutes-1 hour after treatment. The mice dosed at 1 and 2 mL/kg bw, and one animal dosed at 0.5 mL/kg bw, became semi-comatose/comatose, hypothermic and showed laboured breathing within 1 hour. All these animals died within 2-24 hours. The surviving animal dosed at 0.1 and 0.2 mL/kg bw recovered within 18 hours. Apart from the male mouse dosed at 0.5 mL/kg bw, all surviving animals gained weight during the 7 day observation period. Autopsy of the mice that died revealed: Irritation of the stomach and small intestines, dark spleen, congested lungs and mottled liver. Histological examination did not reveal liver necrosis.

In the second study (Jenner, 1964 / Taylor, 1964, rel. 4), the Rat LD50 was calculated to be 1560 mg/kg [1290-1880 mg/kg bw]. Coma was observed soon after treatment. Deaths occurred between 1 hour and 7 days and clinical signs were reported to be scrawny appearance and coma. The Guinea-Pig LD50 was calculated to be 1410 mg/kg [1130-1780 mg/kg bw]. Deaths occurred between 3 and 6 days and clinical signs were reported to be depression and coma.

The lowest LD50 was taken as the key value for this endpoint.

Acute toxicity: dermal

A key study was identified on the registered substance. This acute dermal toxicity study was performed pre-GLP, but was similar to OECD Test Guideline No 402. Five doses were applied undiluted to intact skin of three male and three female rabbits per dose group under occlusion for 24 hours. No mortality was observed at 0.8 mL/kg bw. 1/6, 2/6, 4/6, 6/6 and 6/6 animals died at 1.25, 1.57, 1.98, 3.15 and 5 mL/kg bw, respectively. Thickening of the skin and varying degrees of erythema were observed. Eschar formation developed and necrotic patches began to exfoliate at about Day 7, continuing until termination, and leaving a healthy healing skin under the slough. Systemic toxicity occurred in animals at all doses except 0.8 mL/kg bw. Animals had dyspnea (either slow and laboured or very rapid breathing), nasal discharge (ranging from blood-tinged and frothy to thick and opaque) and congested lungs were found upon necropsy. The incidence and severity of the signs were least in the lower dose groups. All surviving animals showed normal bodyweight gain over the 14-day study period, except for three males (one each from 0.8, 1.25 and 1.98 mL/kg bw group) and one female treated with 0.8 mL/kg bw which showed body weight loss.

Dermal LD50 Combined = 1.77 mL/kg bw (fiducial limits1.35-2.32 mL/kg bw) [equivalent to 1911.6 mg/kg bw (fiducial limits 1458-2505.6 mg/kg bw).

Acute toxicity: inhalation:

No standard acute inhalation toxicity study was available on the registered substance (trans-Isoeugenol). However, relevant data from other inhalation studies are described below:

- A preliminary 3-day exposure study (6 hours/day) was available (WIL, 2012, Rel.2). The aerosolized test material was administered by inhalation to groups of rats (10 animals/sex/dose) at the concentrations of 750 and 300 mg/m3. At 750 mg/m3, the animals were observed to have decreased body weights over the 3-day exposure period (-14 and -10% for males and females, respectively, compared to day 0) and significant clinical observations included rales and laboured respiration in 1/3 males and 2/3 females. At 300 mg/m3, the mean body weight was decreased by 8% and 3 % compared to the study day 0 values for males and females, respectively. At both concentrations, no animals died.

- In the main repeated dose toxicity study conducted according to the OECD Guideline 412 and in compliance with GLP (WIL, 2012, Rel.2), Trans-Isoeugenol was administered by inhalation-aerosol to groups of rats at the concentrations of 1, 10 and 100 mg/m3 for 6 hours per day, 5 days per week for 2 weeks. Epithelial inflammation and degeneration of the nasal cavity were noted in the 1, 10, and 100 mg/m3 group males and females. A dose-relationship was evident as higher incidences and severity of degeneration and subacute inflammation in males and females affecting transitional epithelium (nasal level II) at the 10 and 100 mg/m3 exposure levels, compared with effects at the 1 mg/m3 exposure. Based on severity and combined incidences for males and females, a similar test substance-relationship was evident for subacute inflammation of the respiratory epithelium at nasal level III. The findings were most prominent at nasal levels II and III and showed reduced incidence and severity in the posterior nasal levels (IV-VI), and were generally more frequent in males than in females at all exposure and nasal cavity levels. All nasal cavity findings were minimal to mild in severity and would be considered reversible with removal of the irritant. A LOAEC of 1 mg/m3 for local effects was determined based on the microscopic findings in the nasal tissues at all exposure level.

- In a respiratory lymph node assay (RIVM, 2007, rel.3), groups of mice were exposed to the supporting substance (Isoeugenol, mixture of cis- and trans-Isoeugenol, see Iuclid section 13 for read-across justification) through inhalation, either via vaporization using maximum vapour pressure (11 ppm) or via nebulization in acetone (ca. 300 mg/m3). Mice (6 males per group) were exposed nose-only to test material for 45, 90, 180 or 360 minutes per day on day 0, 1 and 2. Controls were exposed to the vehicle for 360 minutes per day on day 0, 1 and 2. Exposure to aerosols of test material (ca. 300 mg/m3) resulted in toxic effects in the mice that were exposed for 360 min/day. After two days of exposure one mouse died and the other mice displayed several signs of distress. These mice were not exposed to test material on the third day. Effects of the two-day exposure to test material were assessed on day 5. On the third day two mice died that were exposed for 180 minutes/day for 3 days. The other mice in this group appeared normal. This study is not considered reliable for respiratory sensitisation. Indeed, the respiratory lymph node assay is a recently developed animal model and more research is needed to further validate this model (e.g. cut-off value). In addition, in this specific study it is not possible to determine if the level of irritation was excessive or not. However, mortality and clinical data are relevant information to assess the acute toxicity of Isoeugenol.

Even if no LC50 value can be derived from these studies, the data suggest that, in addition to respiratory tract irritation, Trans-Isoeugenol might have an acute toxicity potential at high concentrations. Considering those results together with the acute toxicity hazard identified by oral and dermal routes, the substance is classified as Harmful if inhaled.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

Acute toxicity via Oral route:

Based on the available data on Isoeugenol, Trans-isoeugenol is classified in Category 4 (H302: Harmful if swallowed) according to the Regulation (EC) No. 1272/2008 and of the GHS as the LD50 is below 2000 mg/kg bw (451.5 mg/kg bw).

Acute toxicity via Dermal route:

Based on the available data on Isoeugenol, Trans-Isoeugenol is classified in Category 4 (H312: Harmful in contact with skin) according to the Regulation (EC) No. 1272/2008 and of the GHS as the LD50 is below 2000 mg/kg bw (1911.6 mg/kg bw).

Acute toxicity (Inhalation):

Based on the available data, Trans-Isoeugenol is classified in Category 4 (H332: Harmful if inhaled) according to the Regulation (EC) No. 1272/2008 and of the GHS.

Specific target organ toxicity: single exposure (Oral):

The classification criteria according to the Annex VI of the Regulation (EC) No. 1272/2008 as specific target organ toxicant (STOT) – single exposure, oral are not met since no reversible or irreversible adverse health effects were observed immediately or delayed after exposure and no effects were observed at the guidance value (oral) for a Category 1 classification (C≤ 300 mg/kg bw) and at the guidance value (oral) for a Category 2 classification (2000 mg/kg bw≥C > 300 mg/kg bw). No classification is required.

The criteria for Transient Organ effects (STOT-SE Category 3) according to Annex VI of the Regulation (EC) No. 1272/2008 are not met since narcotic effects were not observed in the acute oral toxicity study.

Specific target organ toxicity: single exposure (Dermal):

The classification criteria according to the Annex VI of the Regulation (EC) No 1272/2008 as specific target organ toxicant (STOT) – single exposure, dermal are not met since no reversible or irreversible adverse health effects were observed immediately or delayed after exposure and no effects were observed at the guidance value (dermal) for a Category 1 classification (C≤ 1000 mg/kg bw) and at the guidance value (dermal) for a Category 2 classification (2000 mg/kg bw≥C > 1000 mg/kg bw). No classification is required.

The criteria for Transient Organ effects (STOT-SE Category 3) according to Annex VI of the Regulation (EC) No. 1272/2008 are not met since narcotic effects were not observed in the acute dermal toxicity study.

Specific target organ toxicity: single exposure (Inhalation):

Based on the available information, Trans-Isoeugenol should be classified as STOT-SE Category 3 (H335: May cause respiratory irritation) according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).