2-isopropyl-N,2,3-trimethylbutyramide

Endpoint summary

Description of key information

A key study (Lackenby (1987)), equivalent or similar to OECD TG 420 was performed to determine the acute oral median lethal dose (LD50) of WS-23 in the rat.

Based on the results of the screening study, eight groups, each of 5 fasted rats (5 males or 5 females) were treated with a single oral dose at dose levels between 255 and 1000 mg/kg.  

The acute oral median lethal dose and 95% fiducial limits were calculated by a probit method. The following values were obtained:

Males (only) - 533 mg/kg (398 - 694)

Females (only) - 490 mg/kg (95% fiducial limits could not be calculated for this sex)

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:

29 January - 25 February 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

OECD 420

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 425 (Acute Oral Toxicity: Up-and-Down Procedure)

Deviations:

yes

Remarks:

One animal was outside the pre-fasted body weight range quoted in the protocol. This deviation was considered not to have affected the integrity of the study.

GLP compliance:

yes

Test type:

up-and-down procedure

Limit test:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: WS23, white powder, S15100-T01


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: all formulations used within 4 hours of preparation and assumed to be stable unless specified by study sponsor. The test article was formulated in corn oil.

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Females non-pregnant: yes
- Age at study initiation: the animals were aged between 6 and 10 weeks
- Weight at study initiation: the males were between 263 and 363 g. The females were between 170 and 275 g.
- Fasting period before study: overnight fasting before dosing
- Housing: groups in 5 in stainless steel wire mesh cages cages
- Diet (e.g. ad libitum): except the overnight fasting before dosing, animals were allowed free access to food. The food was re-introduced 1-hr after treatment.
- Water (e.g. ad libitum): yes - tap water via bottles ad libitum
- Acclimation period: minimum 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 40-70%
- Air changes (per hr): minimum 15 air changes per hour
- Photoperiod (hrs dark / hrs light): fluorescent, 12 hours each day (between 6:00 AM - 18:00 PM)

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

VEHICLE
- Vehicle: corn oil
- Amount of vehicle (if gavage): 10 ml/kg

MAXIMUM DOSE VOLUME APPLIED: 10 ml/kg

Doses:

Screening study - doses range between 250 and 5000 mg/kg
Definitive study - doses range between 255 and 1000 mg/kg

No. of animals per sex per dose:

Screeenign study - 1 male and 1 female per single oral dose
Definitive study - 5 males and 5 females per single oral dose

Control animals:

no

Details on study design:

Experimental design:

Screening study - groups of one male and one female rat were dosed with 1 of 5 appropriately spaced dose levels, with 5000 mg/kg as the highest dose.
Animals were observed for 7 days. The mortality was recorded.

As the deaths were noted in the screening study, a definitive study was applied

Definitive study - five females and five males were used per dose. Dose levels were based on results from the screening study.

- Clinical observations: 1 and 4 hours after dosing, then daily for 14 days
- Body weight observation: day before treatment (day 1), day of dosing, days 8 and 15 and at death
- Necropsy of survivors was performed. All animals were subjected to a gross necropsy examination.
No tissues were retained. Animals surviving the 14 day observation period were killed by exposure to high levels of carbon dioxide.
- Other examinations performed: clinical signs, body weight

Statistics:

The acute oral median lethal dose and 95% fiducial limits for all animals was calculated using a probit analysis (Finney, D.J. (1971), Probit Analysis, 3rd ed. Cambridge: Cambridge University Press.

Preliminary study:

During the screening study 5 groups of animals, each of 2 animals (1 male and 1 female) were dosed with 5000, 2000,1000, 500 and 250 mg/kg. The mortalities indicated an oral median lethal dose between 250 and 1000 mg/kg.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

490 mg/kg bw

Based on:

other:

Remarks on result:

other:

Remarks:

95% fiducial limits cannot be determined

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

533 mg/kg bw

Based on:

other:

95% CL:

ca. 533 - ca. 694

Mortality:

The animals were observed for mortality only during the screening study. The mortalities indicated an oral median lethal dose between 250 and 1000 mg/kg.
During the definitive study, a total of 20 animals (12 male, 8 female) died during the study period.

Definitive study - 2 animals treated with 1000 mg/kg were found dead 4 hours after treatment. The majority of animals were found dead 1 day after treatment. Deaths continued until 3 days after treatment.

Clinical signs:

other: Definitive study - marked clinical signs were observed in all male animals treated at 360 to 1000 mg/kg during the day of treatment. Thereafter fewer clinical signs were observed until day 5 when all surviving animals treated at 510 mg/kg were normal. Two

Gross pathology:

All animals necropsied at termination were unremarkable.

Necropsy of animals dying during the study showed abnormal appearance of the lungs, liver, kidneys, stomach, G.I. tract and bladder. Common findings in these animals were dark lungs, dark liver, distended stomach, discoloured G.I. tract and distended bladder.

Table 3. Mortalities in the screening study
Group number	Dose Level (mg/Kg)	Male mortalities	Female mortalities	Total	%
1	5000	1/1	1/1	2/2	100
2	2000	1/1	1/1	2/2	100
3	1000	1/1	1/1	2/2	100
4	500	0/1	1/1	1/2	50
5	250	0/1	0/1	0/2	0

Table 3. Mortalities in the definitive study
Group number	Dose Level (mg/Kg)	Male mortalities	Female mortalities	%
6	360	0/5	-	0
7	510	3/5	-	60
8	720	4/5	-	80
9	1000	5/5	-	100
10	255	-	0/5	0
11	360	-	0/5	0
12	500	-	3/5	60
13	700	-	5/5	100

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

This study was performed to determine the acute oral median lethal dose (LD50) of WS-23 in the rat. The acute oral median lethal dose and 95% fiducial limits were calculated by a probit method. The following values were obtained:
Males (only) - 533 mg/kg (398 - 694)
Females (only) - 490 mg/kg (95% fiducial limits could not be calculated for this sex)

Based on these results, WS-23 is classified as acute oral toxicity cat. 4 according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Executive summary:

This study was performed to determine the acute oral median lethal dose (LD50) of WS23 in the rat.

The animals were aged between 6 and 10 weeks and were acclimatised to the laboratory environment for at least 3 days. Before starting the study, all animals were examined for signs of ill health or injury. All animals appeared healthy and no animals were discarded.

In the screening study, 5 groups of animals, each of 2 animals (1 male, 1 female) were dosed with 5000, 2000,1000, 500 and 250 mg/kg. The animals were observed for mortality only in this phase of the study. Frequent observations were made for 7 days. Individual body weights were recorded on the day of treatment to allow the calculation of individual treatment volumes. No necropsies were performed. The mortalities indicated an oral median lethal dose between 250 and 1000 mg/kg.

Based on the results of the screening study, 8 groups, each of 5 fasted rats (5 males or 5 females) were treated with a single oral dose at dose levels between 255 and 1000 mg/kg. All animals were observed for overt signs of toxicity or behavioural change at 1  and 4 hours after treatment and subsequently once daily for 14 days. All gross or visible toxic or pharmacological effects were recorded. Marked clinical signs were observed in a majority of female animals treated at 255 to 700 mg/kg during the day of treatment. Thereafter fewer clinical signs were observed until day 3 when all animals treated at 255 and 360 mg/kg were normal. The 2 surviving animals treated at 500 mg/kg were normal on day 5.

The acute oral median lethal dose and 95% fiducial limits were calculated by a probit method. The following values were obtained:

Males (only) - 533 mg/kg (398 - 694)

Females (only) - 490 mg/kg (95% fiducial limits could not be calculated for this sex)

Based on these results, WS-23 is classified as acute oral toxicity cat. 4 according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

490 mg/kg bw

Justification for classification or non-classification

Based on the available data of acute oral toxicity, WS-23 is classified as acute oral toxic category 4 according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.