Reaction mass of methyl 2-[[(E)-(2,4-dimethylcyclohex-3-en-1-ylidene)methyl]amino]benzoate and methyl 2-[[(Z)-(2,4-dimethylcyclohex-3-en-1-ylidene)methyl]amino]benzoate

Endpoint summary

Description of key information

The acute oral toxicity of the test item was determined by the Acute Toxic Class Method in female Sprague-Dawley rats. Three animals were administered a single oral dose of 5000 mg/kg body weight (bw) by intubation. Mortality, clinical signs of toxicity and body weight were observed for 14 days following administration. Mortality was not observed at any tested dose level, and the LD50 was expected to be >5000 mg/kg bw. Chromaturia, compound-coloured stool, decrease in locomotor activity, decrease of faecal volume, mucous stool, soiled perineal region, abnormal gait and alterations in body weight observed on Days 1 to 3 were considered test substance related. Conducted according to OECD 423, GLP and considered reliable without restriction (Klimisch 1), there is no evidence of a relevant intrinsic acute oral toxicity requiring classification or substance specific risk mitigation measures (RMM).

 

The acute dermal toxicity of the test item was determined in a limit test with male and female Sprague-Dawley rats (2000). There were no mortalities observed over the 14-day observation period, therefore the LD50 was determined to be >2000 mg/kg bodyweight. No signs of dermal irritation, clinical signs of toxicity or abnormal bodyweight changes were observed throughout the study duration. Conducted according to OECD 402, GLP and considered reliable without restriction (Klimisch 1), there is no evidence of a relevant intrinsic acute dermal toxicity requiring classification or substance specific RMM. 

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 April 2016 - 25 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

An assessment of acute toxicity is required to fulfil REACH Annex VII information requirements. The acute oral toxicity study was conducted according to OECD TG 423, GLP and is considered reliable without restriction (Klimisch 1).

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Version / remarks:

2001

Deviations:

no

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species, strain: Sprague-Dawley (Crl:CD(SD)), SPF
- Age at study initiation: 8 weeks
- Weight at study initiation: 179.9-190.2 g
- Fasting period before study: Approximately 16 hours
- Housing: Housed individually in stainless wire mesh cages (260 x 350 x 210 mm)
- Diet (e.g. ad libitum): Pelleted rodent chow, ad libitum
- Water (e.g. ad libitum): Public tap water in Cheongju-si was filtered and irradiated by ultraviolet light and provided ad libitum.
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1−23.2°C
- Humidity (%): 45.1−54.2%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle, 150-300 Lux

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 500 mg/mL
- Lot/batch no. (if required): MKBV2080V

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg

DOSAGE PREPARATION (if unusual): The test item was weighed into a bottle and a small amount of corn oil was added. The test item was dissolved using a vortex mixer. Corn oil was gradually added to yield the desired concentration.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: Due to the low expected toxicity of the test substance, based on information supplied by the sponsor, a starting dose of 5,000 mg/kg was administered in one animal for this study (Step 1).

Doses:

5000 mg/kg bodyweight

No. of animals per sex per dose:

1 for Step 1, 2 for Step 2

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: All animals were observed for mortality, general condition and clinical signs (type, severity, time of onset and recovery) at 30 minutes after dosing and at 1, 2, 4 and 6 hours after dosing on Day 0 and once daily thereafter for 14 days (Day 1−Day 14). The body weight was recorded prior to dosing on Day 0 and on Days 1, 3, 7 and on the day of necropsy, Day 14.
- Necropsy of survivors performed: Yes
- Other examinations performed: clinical signs and body weight

Statistics:

Statistical analysis was not performed. Mean scores and values are determined.

Key result

Sex:

female

Dose descriptor:

LD50 cut-off

Effect level:

5 000 mg/kg bw

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Mortality:

All animals at 5,000 mg/kg survived the duration of the study. There were no effects on mortality.

Clinical signs:

other: Chromaturia (abnormal colour of the urine) were observed in all animals at 6 hours after dosing at 5,000 mg/kg. Chromaturia, compound-coloured stool, decrease in locomotor activity, decrease of faecal volume, mucous stool, soiled perineal region and/or ab

Gross pathology:

No abnormal morphological findings were observed in any animal at 5,000 mg/kg.

Interpretation of results:

GHS criteria not met

Conclusions:

In an acute oral toxicity study conducted according to OECD TG 423, GLP and considered reliable without restriction (Klimisch 1), the LD50 (cut-off) was determined to be ≥ 5,000 mg/kg bw.

Executive summary:

The acute oral toxicity of the test item was determined by the Acute Toxic Class Method in female Sprague-Dawley rats. Three animals were administered a single oral dose of 5000 mg/kg body weight (bw) by intubation. Mortality, clinical signs of toxicity and body weight were observed for 14 days following administration. Mortality was not observed at any tested dose level, and the LD50 was expected to be >5000 mg/kg bw. Effects considered to be test substance related included chromaturia, compound-colored stool, decrease in locomotor activity, decrease of fecal volume, mucous stool, soiled perineal region, abnormal gait, suppression of body weight gain and/or decreased bodyweights on Days 1 and 2.

 

This study is considered to be reliable without restrictions (Klimisch 1) as it was GLP-compliant and was performed according to OECD guideline 423. There is no evidence of a relevant intrinsic acute oral toxicity requiring classification or substance specific risk mitigation measures (RMM).

Reference 2

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1 July 1981

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

Summary page of experimental details and results only.

Justification for type of information:

An assessment of acute toxicity is required to fulfil REACH Annex VII information requirements. Whilst the study was not considered reliable, due to insufficient reporting, in accordance with Annex VI, all existing information should be evaluated prior to the conduct of in vivo testing.

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: A single dose of the test item was administered by oral gavage. Test animals were observed for 14 days following exposure.
- Short description of test conditions: Rats were administered the test item (3.16, 4.64, 6.81, 8.25 or 10.00 ml/kg bw) in a constant volume of 20 mL/kg body weight of the test item in 0.8% aqueous hydroxypropyl-methylcellulose gel.
- Parameters analysed / observed: Number of dead animals, clinical signs of toxicity

GLP compliance:

no

Test type:

fixed dose procedure

Limit test:

no

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

Standard conditions

Route of administration:

oral: gavage

Vehicle:

other: 0.8% hydroxypropyl-methylcellulose gel

Details on oral exposure:

MAXIMUM DOSE VOLUME APPLIED: 20 mL/kg bodyweight

Doses:

3.16, 4.64, 6.81, 8.25, 10.00 mL/kg

No. of animals per sex per dose:

2

Control animals:

not specified

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Not reported
- Necropsy of survivors performed: No
- Other examinations performed: Clinical signs and pathology

Statistics:

The LD50 was determined graphically.

Sex:

male

Dose descriptor:

LD100

Effect level:

10 mL/kg bw

Based on:

test mat.

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

8.25 mL/kg bw

Based on:

test mat.

Sex:

male

Dose descriptor:

LD0

Effect level:

6.81 mL/kg bw

Based on:

test mat.

Sex:

male

Dose descriptor:

LD0

Effect level:

4.64 mL/kg bw

Based on:

test mat.

Sex:

male

Dose descriptor:

LD0

Effect level:

3.16 mL/kg bw

Based on:

test mat.

Mortality:

One animal died when dosed at 8.25 mL/kg bodyweight. Two animals died when dosed at 10.00 mL/kg bodyweight.

Clinical signs:

other: - Duration of observation period following administration: 14 days - Frequency of observations and weighing: Not reported - Necropsy of survivors performed: No - Other examinations performed: Clinical signs and pathology - Decreased motility - Ataxia - Un

Gross pathology:

Liver and kidneys were pale.

Table 1. Mortality results

Test item dose (mL/kg)	Number of animals tested	Number of dead animals
3.16	2	0
4.64	2	0
6.81	2	0
8.25	2	1
10.00	2	2

Interpretation of results:

study cannot be used for classification

Conclusions:

The LD50 of the test item was graphically determined to be 8.25 mL/kg bodyweight.

Executive summary:

The acute oral toxicity of the test item was determined in a 14-day study. Male mice were given a single dose of the test item in 0.8% hydroxypropyl-methylcellulose gel by oral intubation. Doses were administered at a constant volume of 20 mL/kg bodyweight at test item concentrations of 3.16, 4.64. 6.8, 8.25 and 10.00 mL/kg bw. Each treatment was tested in duplicate. The LD50 of the test item was graphically determined to be 8.25 mL/kg bw. Mice showed signs of decreased motility, ataxia, unusual positions, dyspnoea, lacrimation and tonic spasms. The reliability of this study was not assignable (Klimisch 4), as only a summary page of experimental protocols and results is available. The study was not considered sufficient to fulfil REACH Annex VII information requirements.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating dose

Value:

5 000 mg/kg bw

Quality of whole database:

Two acute oral toxicity studies are available (1981, 2016). The key study is considered to be reliable without restrictions (Klimisch 1) as it was GLP-compliant and was performed according to OECD guideline 423. The reliability of supporting study cannot be assigned (Klimisch 4), due to insufficient reporting.

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:

10 April 2000 - 24 April 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

An assessment of acute toxicity is required to fulfil REACH Annex VII information requirements. The acute dermal toxicity study was conducted according to OECD TG 402, GLP and is considered reliable without restriction (Klimisch 1).

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Version / remarks:

1987

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species, Strain: Sprague-Dawley CD (Crl:CD (SD) IGS BR)
- Age at study initiation: 8-12 weeks old
- Weight at study initiation: Males - 200-218 g. Females - 201-221 g
- Housing: Suspended polypropylene cages furnished with woodflakes
- Diet: Rat and Mouse Expanded Diet No.1, ad libitum
- Water: Mains drinking water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

TEST SITE
- Area of exposure: Back and flanks
- % coverage: 10
- Type of wrap if used: Surgical gauze semi-occluded with a piece of self-adhensive bandage

REMOVAL OF TEST SUBSTANCE
- Washing: Wiped with cotton wool moistened with arachis oil BP
- Time after start of exposure: 24 hours

TEST MATERIAL
- Concentration: 2000 mg/kg bodyweight

Duration of exposure:

24 hour exposure, with a 14-day observation period

Doses:

2000 mg/kg bodyweight

No. of animals per sex per dose:

5 males and 5 females

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: 0.5, 1, 2 and 4 hours after dosing, then once daily for 14 days
- Necropsy of survivors performed: yes
- Other examinations performed: Mortality, clinical signs, dermal reactions, body weight

Key result

Sex:

male/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 deaths

Clinical signs:

other: No clinical signs of toxicity were noted during the study.

Gross pathology:

No abnormalities were noted at necropsy.

Other findings:

No signs of dermal Irritation were noted during the study.

Interpretation of results:

GHS criteria not met

Conclusions:

The LD50 of the test item was determined to be > 2000 mg/kg bodyweight.

Executive summary:

The acute dermal toxicity of the test item was determined in a limit test with male and female Sprague-Dawley rat. The backs and flanks of the animals were shorn and the test material was applied unchanged at a dosage of 2000 mg/kg bodyweight. The treatment sites were semi-occluded and rats were exposed to the test item for 24 hours. Mortality, clinical signs of toxicity, dermal reactions and body weight were observed for 14 days following treatment. There were no mortalities observed, therefore the LD50 was determined to be > 2000 mg/kg bodyweight. No signs of dermal irritation, clinical signs of toxicity or abnormal bodyweight changes were observed throughout the study duration. This study is considered to be reliable without restrictions (Klimisch 1) as it was GLP-compliant and was performed according to OECD guideline 402.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating dose

Value:

2 000 mg/kg bw

Quality of whole database:

One acute dermal toxicity studies is available (2000). This study is considered to be reliable without restrictions (Klimisch 1) as it was GLP-compliant and was performed according to OECD guideline 402.

Additional information

The acute oral toxicity of the test item was determined by the Acute Toxic Class Method using female Sprague-Dawley rats (2016). One test animal in Step 1 and two test animals in Step 2 were given a single dose of the test item at a concentration of 5000 mg/kg bodyweight by oral intubation. Mortality, clinical signs of toxicity and body weight were observed for 14 days following administration. There were no mortalities observed, therefore the LD50 (cut-off) was determined to be ≥5000 mg/kg b.w. This study is considered to be reliable without restrictions (Klimisch 1) as it was GLP-compliant and was performed according to OECD guideline 423, and is therefore sufficient to meet the Annex VII information requirement.

 

Another 14 -day acute oral toxicity study is available as supporting data (1981). Male mice were given a single dose of the test item in 0.8% hydroxypropyl-methylcellulose gel by oral intubation. Doses were administered at a constant volume of 20 mL/kg bodyweight at test item concentrations of 3.16, 4.64. 6.8, 8.25 and 10.00 mL/kg. Each treatment was tested in duplicate. The LD50 of the test item was graphically determined to be 8.25 mL/kg bodyweight. Rats showed signs of decreased motility, ataxia, unusual positions, dyspnea, lacrimation and tonic spasms. The reliability of this study is not assignable (Klimisch 4) as only a summary page of experimental details and results is available, however it is suitable for use as supporting data to meet the Annex VII information requirement.

 

The acute dermal toxicity of the test item was determined in a limit test with male and female Sprague-Dawley rats (2000). The backs and flanks of the animals were shorn and the test material was applied unchanged at a dosage of 2000 mg/kg bodyweight. The treatment sites were semi-occluded and rats were exposed to the test item for 24 hours. Mortality, clinical signs of toxicity, dermal reactions and body weight were observed for 14 days following treatment. There were no mortalities observed, therefore the LD50 was determined to be >2000 mg/kg bodyweight. No signs of dermal irritation, clinical signs of toxicity or abnormal bodyweight changes were observed throughout the study duration. This study is considered to be reliable without restrictions (Klimisch 1) as it was GLP-compliant and was performed according to OECD guideline 402, and is therefore sufficient to meet the Annex VII information requirement.

Justification for classification or non-classification

Two key studies (Klimisch 1) are available for acute toxicity classification (2000, 2016). The oral LD50 was determined to be ≥5000 mg/kg bw using the acute toxic class method and the dermal LD50 was determined to be >2000 mg/kg bw in a limit test. Therefore, according to CLP Regulation (EC) No. 1272/2008, the test item should not be classified for acute oral toxicity.