methyl 2-hydroxybut-3-enoate

Endpoint summary

Description of key information

The tests were conducted with the purified and stabilised (registered) form of the test substance. 

 

In an acute oral toxicity test according to OECD guideline 423, the LD50 of the test item was found to be between 300 and 2000 mg/kg bw (LD50 cut-off: 500 mg/kg bw).

 

In an acute dermal toxicity study according to OECD guideline 402 and GLP, the test item could not be classified into any toxicity category as delayed local corrosive effects occured.

 

On this basis, the test item was classified as corrosive to skin and further acute dermal or inhalation toxicity testing was waived.

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:

2020-05-13 to 2020-06-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Version / remarks:

2008-05-30

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2001-12-17

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Han: WIST

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: TOXI COOP ZRT., Cserkesz u. 90. 1103 Budapest, Hungary
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Young adult rats, 8-9 weeks old in first, second and third step
- Weight at study initiation: 181-184 g (first step), 183-184 g (second step), 180-184 g (third step)
- Fasting period before study: The day before treatment, the animals were fasted. The food but not water was withheld overnight. The food was given back 3 hours after the treatment.
- Housing: Group caging (3 animals/cage) in type III polypropylene/polycarbonate cages with laboratory bedding
- Diet: Ad libitum (ssniff® SM R/M-Z+H complete diet for rats and mice)
- Water: Ad libitum (tap water)
- Acclimation period: 6 days in first step, 7 days in second step and 8 days in third step
- Microbiological status when known: SPF at arrival, good conventional during the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70 %
- Air changes (per hr): More than 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From day 1 to day 15

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 30 mg/mL (for 300 mg/kg bw treatment) and 200 mg/mL (for 2000 mg/kg bw treatment)
- Amount of vehicle: 10 mL/kg bw
- Justification for choice of vehicle: Water is the preferred vehicle according to OECD guideline 423.

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw

DOSAGE PREPARATION: Formulations were prepared just before the administration and were stirred continuously during the treatment.

Doses:

300 and 2000 mg/kg bw

No. of animals per sex per dose:

Three female animals for step 1 (300 mg/kg bw), 2 (300 mg/kg bw) and 3 (2000 mg/kg bw) respectively

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Animals were observed individually after dosing at least once during the first 30 minutes, then 1 h, 2 h, 3 h, 4 h after the treatment and twice each day for 14 days thereafter. The body weights were recorded on day 0 (just before the treatment), on day 1, on day 7 and on day 15.
- Necropsy of survivors performed: Yes
- Clinical signs including body weight: Individual observations were performed on the skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Statistics:

Not required

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

500 mg/kg bw

Based on:

test mat.

Remarks on result:

other: LD cut-off according to OECD guideline 423

Mortality:

Step 1 (300 mg/kg bw): No mortality.
Step 2 (300 mg/kg bw): 1/3 animals died on day 1.
Step 3 (2000 mg/kg bw): 3/3 animals died within 30 minutes after the treatment.

Clinical signs:

convulsions

irregular respiration

lethargy (hypoactivity)

salivation

Body weight:

lower than 10% body weight loss

Gross pathology:

Step 2 (300 mg/kg bw): Cannibalised left thigh, stomach full of gas, frothy discharge in the stomach, red and haemorrhaged mucous membrane in the stomach in the animals that died.

Step 3 (2000 mg/kg bw): Saliva around the mouth in all animals

Other findings:

- Potential target organs: The observed necropsy findings at 300 mg/kg bw indicated an irritation effect in the stomach which might have caused the death in the one animal of step 2. No such changes were noted in the step 3 animals, most likely to the early death within 30 min of the administration.

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

In an acute oral toxicity test according to OECD guideline 423, the LD50 of the test item was found to be between 300 and 2000 mg/kg bw (LD50 cut-off: 500 mg/kg bw).

Executive summary:

In an acute oral toxicity study according to OECD guideline 423 and GLP, the acute toxic class method was used involving a stepwise procedure with the use of 300 mg/kg bw as the starting dose in three female rats. No animal died in the first group, so further three female rats were treated with the same dose (300 mg/kg bw). Only one animal died in the second group, so further three female rats were treated with 2000 mg/kg bw. All animals died in third group, so the test was finished as the stopping criteria of Annex 2c of OECD Guideline No. 423 (presented in Appendix VII) were met. Animals were weighed, observed for lethality and toxic symptoms for 14 days after the treatment. Gross pathological examination was carried out in animals died on the treatment day or on Day 1, as well as 15th day after the treatment in surviving animals.

 

All female rats treated with the test item survived until the end of the 14-day observation period in step 1. One rat in step 2 died on Day 1. All rats dosed at 2000 mg/kg bw died on the treatment day 30 minutes after the treatment. In the first step, symptoms like decreased activity, bedding chewing and disturbance of the autonomic functions (salivation) were observed on the treatment day 30 minutes after the administration. The bedding chewing might be caused by stomach mucous membrane irritation effects of the test item, the pathological finding noted in the animal that died on day one in group two. In the second step, a symptoms like decreased activity and bedding chewing and disturbance of the autonomic functions (salivation) were observed on the treatment day between 30 minutes and 1 hour after the treatment. The bedding chewing might be caused by stomach mucous membrane irritation effects of the test item the pathological findings noted in the animal that died on day one in this group. In the third step, CNS symptoms (tonic convulsion, clonic convulsion), disturbances of coordination (prone position, incoordination) and disturbances of the autonomic functions (lacrimation, salivation, dyspnoea) were observed on the treatment day 30 minutes after the treatment. These symptoms were connected with massive systemic toxic effect of the test item. The body weight development was undisturbed in all surviving animals.

 

One of six animals treated with 300 mg/kg bw died spontaneously during the study and was necropsied on Day 1. Five animals of the same dose survived until the scheduled autopsy on Day 15. All of three animals treated with 2000 mg/kg bw died spontaneously and within 30 min during the study and were necropsied on the treatment day. External necropsy finding as cannibalised left thigh was observed in the one dead animal of the 300 mg/kg bw dose group. Internal necropsy findings were recorded in the stomach of same animal as follows: gas- and frothy discharge content and red and haemorrhaged mucous membrane. These internal findings showed that the test item is causing stomach mucous membrane irritation effects. External necropsy finding as saliva around the mouth was observed in animals treated with 2000 mg/kg bw. No other specific findings at internal necropsy were noted, most likely due to the early onset of lethality. All organs of the surviving animals proved to be free of treatment related gross pathological changes.

 

The test item was ranked into classes of Globally Harmonized Classification System (GHS) described in the OECD Guideline No. 423 as below:

 

Dose (mg/kg bw)	Mortality (dead/treated)	LD50 (mg/kg bw)	GHS category
300	1/6	500	4
2000	3/3

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

500 mg/kg bw

Quality of whole database:

Reliable without restrictions

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:

2021-09-20 to 2021-11-08

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Short report

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-05-30

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1200 (Acute Dermal Toxicity)

Version / remarks:

1998-08

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity: Fixed Dose Procedure)

Version / remarks:

2017-10-09

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

fixed dose procedure

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Han:WIST

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. Cserkesz u. 90., 1103 Budapest, Hungary
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Young adult rats, min. 8-10 weeks
- Weight at study initiation: 200-300 g
- Fasting period before study: No
- Housing: Max. 3 animals/cage during acclimatisation and individually during exposure in type III polypropylene/polycarbonate cages with certified laboratory bedding
- Diet: Ad libitum (ssniff® SM R/M-Z+H complete diet)
- Water: Ad libitum (tap water)
- Acclimation period: At least 5 days
- Microbiological status: SPF at arrival and kept in a good conventional environment during the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): More than 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From day 0 to day 14

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

TEST SITE
- Area of exposure: 10 % area of the total body surface
- % coverage: 10 % area of the total body surface
- Type of wrap: Sterile gauze pads were kept in contact with the skin by a patch with adhesive hypoallergenic plaster. The entire trunk of the animal will then be wrapped with semi-occlusive plastic wrap.

REMOVAL OF TEST SUBSTANCE
- Washing: Residual test item was removed, using water at body temperature.
- Time after start of exposure: 24 hours

Duration of exposure:

24 hours

Doses:

200, 1000 and 2000 mg/kg bw

No. of animals per sex per dose:

One female animal per dose (200, 1000 and 2000 mg/kg bw) was treated with the test item in the range-finding test and one female animal was treated with a single dose (200 mg/kg bw) in the main test.

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Animals were weighted directly before treatment and at day 7 and 15 or at preliminary sacrifice
- Necropsy of survivors performed: Yes

Statistics:

Not required

Preliminary study:

A range-finding study was performed to determine the starting dose for the main test. The starting dose of the range-finding study was 200 mg/kg bw. The animal did not die in this first step, therefore one further rat was treated with 1000 mg/kg body weight in range-finding study. This animal did not die in second step, therefore one further rat was treated with 2000 mg/kg body weight in range-finding study. This animal died on Day 1. The animal treated with 1000 mg/kg bw was however humanely killed on Day 5 as the test item caused with some delay corrosive local signs.

Key result

Sex:

female

Dose descriptor:

LD50

Remarks on result:

not determinable

Remarks:

Not determinable as the substance induced delayed local corrosive effects and the study was stopped for animal welfare reasons.

Mortality:

Yes, the animal treated with 2000 mg/kg bw died on day 1. Animals treated with 1000 and 200 (main test) mg/kg bw were humanely killed before the end of the 14-day observation period as the test item caused with some delay corrosive local signs.

Clinical signs:

other:

Body weight:

other body weight observations

Remarks:

The body weight was only measured for the rat dosed with 200 mg/kg bw in the range-finding test as all other animals had to be humanely killed before further weighting was possible. The rat dosed with 200 mg/kg bw showed a stable body weight gain.

Gross pathology:

Not performed

Other findings:

The test item induced local corrosive symptoms like erythema, oedema, crust, (bloody) wounds, scabs and necrosis

Interpretation of results:

study cannot be used for classification

Conclusions:

In an acute dermal toxicity study according to OECD guideline 402 and GLP, the test item could not be classified into any toxicity category as delayed local corrosive effects occured.

Executive summary:

In an acute dermal toxicity study according to OECD guideline 402 and GLP, the toxicity of test item was assessed when administered in a single dermal dose to rats at one or more defined dose levels. At first, the range-finding study was performed in one female Han:WIST rat. The starting dose was 200 mg/kg bw. The test item was applied in original form and left in contact with the skin for a 24 hours period in all animals. The animal did not die in this first step, therefore one further rat was treated with 1000 mg/kg body weight in range-finding study. This animal did not die in second step, therefore one further rat was treated with 2000 mg/kg body weight in range-finding study. This animal died on Day 1. The animal treated with 1000 mg/kg bw was however humanely killed on Day 5 as the test item caused with some delay corrosive local signs. Based on this finding one further animal was treated 200 mg/kg bw in main study. This animal was however also humanely killed on Day 4 because of massive skin corrosion developing with time. The study was terminated for animal welfare reasons, because the test item caused massive skin damage up to clear and massive skin corrosion over time at 1000 mg/kg bw, and even at 200 mg/kg bw in the second test animal. This massive skin damage was not to be expected based on the in vitro skin irritation/corrosivity tests classifying the test item as irritant but clearly not as corrosive.

Additional information

Acute oral toxicity, rat, RL1

In an acute oral toxicity study according to OECD guideline 423 and GLP, the acute toxic class method was used involving a stepwise procedure with the use of 300 mg/kg bw as the starting dose in three female rats. No animal died in the first group, so further three female rats were treated with the same dose (300 mg/kg bw). Only one animal died in the second group, so further three female rats were treated with 2000 mg/kg bw. All animals died in third group, so the test was finished as the stopping criteria of Annex 2c of OECD Guideline No. 423 (presented in Appendix VII) were met. Animals were weighed, observed for lethality and toxic symptoms for 14 days after the treatment. Gross pathological examination was carried out in animals died on the treatment day or on Day 1, as well as 15th day after the treatment in surviving animals.

 

All female rats treated with the test item survived until the end of the 14-day observation period in step 1. One rat in step 2 died on Day 1. All rats dosed at 2000 mg/kg bw died on the treatment day 30 minutes after the treatment. In the first step, symptoms like decreased activity, bedding chewing and disturbance of the autonomic functions (salivation) were observed on the treatment day 30 minutes after the administration. The bedding chewing might be caused by stomach mucous membrane irritation effects of the test item, the pathological finding noted in the animal that died on day one in group two. In the second step, a symptoms like decreased activity and bedding chewing and disturbance of the autonomic functions (salivation) were observed on the treatment day between 30 minutes and 1 hour after the treatment. The bedding chewing might be caused by stomach mucous membrane irritation effects of the test item the pathological findings noted in the animal that died on day one in this group. In the third step, CNS symptoms (tonic convulsion, clonic convulsion), disturbances of coordination (prone position, incoordination) and disturbances of the autonomic functions (lacrimation, salivation, dyspnoea) were observed on the treatment day 30 minutes after the treatment. These symptoms were connected with massive systemic toxic effect of the test item. The body weight development was undisturbed in all surviving animals.

 

One of six animals treated with 300 mg/kg bw died spontaneously during the study and was necropsied on Day 1. Five animals of the same dose survived until the scheduled autopsy on Day 15. All of three animals treated with 2000 mg/kg bw died spontaneously and within 30 min during the study and were necropsied on the treatment day. External necropsy finding as cannibalised left thigh was observed in the one dead animal of the 300 mg/kg bw dose group. Internal necropsy findings were recorded in the stomach of same animal as follows: gas- and frothy discharge content and red and haemorrhaged mucous membrane. These internal findings showed that the test item is causing stomach mucous membrane irritation effects. External necropsy finding as saliva around the mouth was observed in animals treated with 2000 mg/kg bw. No other specific findings at internal necropsy were noted, most likely due to the early onset of lethality. All organs of the surviving animals proved to be free of treatment related gross pathological changes.

 

The test item was ranked into classes of Globally Harmonized Classification System (GHS) described in the OECD Guideline No. 423 as below:

Dose (mg/kg bw)	Mortality (dead/treated)	LD50 (mg/kg bw)	GHS category
300	1/6	500	4
2000	3/3

 

Acute dermal toxicity, RL2

The objective of the study was to assess the toxicity of test item when administered in a single dermal dose to rats at one or more defined dose levels. At first, the range-finding study was performed in one female Han:WIST rat. The starting dose was 200 mg/kg bw. The test item was applied in original form and left in contact with the skin for a 24 hours period in all animals. The animal did not die in this first step, therefore one further rat was treated with 1000 mg/kg body weight in range-finding study. This animal did not die in second step, therefore one further rat was treated with 2000 mg/kg body weight in range-finding study. This animal died on Day 1. The animal treated with 1000 mg/kg bw was however humanely killed on Day 5 as the test item caused with some delay corrosive local signs. Based on this finding one further animal was treated 200 mg/kg bw in main study. This animal was however also humanely killed on Day 4 because of massive skin corrosion developing with time. The study was terminated for animal welfare reasons, because the test item caused massive skin damage up to clear and massive skin corrosion over time at 1000 mg/kg bw, and even at 200 mg/kg bw in the second test animal. This massive skin damage was not to be expected based on the in vitro skin irritation/corrosivity tests classifying the test item as irritant but clearly not as corrosive.

Justification for classification or non-classification

The available experimental test data on acute oral toxicity are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data, the test item is classified and labelled for acute oral toxicity (Category 4) according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.

 

Based on its corrosiveness, the test substance cannot be classified or labelled for acute dermal toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.