methyl 2-hydroxybut-3-enoate

Acute Toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-05-30

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

methyl 2-hydroxybut-3-enoate

EC Number:

831-109-9

Cas Number:

5837-73-0

Molecular formula:

C5H8O3

IUPAC Name:

methyl 2-hydroxybut-3-enoate

Test animals

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

Administration / exposure

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

Results and discussion

Effect levels

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.

Applicant's summary and conclusion

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