Methyl acetoacetate

Acute Toxicity: dermal

Administrative data

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 30 January 2012 and 13 February 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: study report was conclusive, done to a valid guideline and conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Methyl acetoacetate (MAA)

IUPAC Name:

Methyl acetoacetate (MAA)

Test material form:

other: liquid (undiluted)

Details on test material:

- Sponsor's identification: Methyl acetoacetate (MAA)
- Description: clear colourless liquid
- Date received: 05 December 2011
- Expiry date: 28 November 2013
- Storage conditions: room temperature in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Five male and five female Wistar (RccHan:WIST) strain rats were supplied by Harlan Laboratories UK Ltd., Oxon, UK. On receipt the animals were randomly allocated to cages. The females were nulliparous and non pregnant. After an acclimatisation period of at least five days the animals were selected at random and given a number unique within the study by indelible ink-marking on the tail and a number written on a cage card. At the start of the study the animals weighed at least 200g, and were eight to twelve weeks of age. The weight variation did not exceed ±20% of the mean weight for each sex.
The animals were housed in suspended solid floor polypropylene cages furnished with woodflakes. The animals were housed individually during the 24 Hour exposure period and in groups of five, by sex, for the remainder of the study. Free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analysed and were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 19 to 25°C and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

The calculated volume of test item, as received, was applied as evenly as possible to an area of shorn skin (approximately 10% of the total body surface area) using a graduated syringe.

Duration of exposure:

24 hours

Doses:

2000 mg /kg body weight

No. of animals per sex per dose:

5 males & 5 females

Control animals:

not required

Details on study design:

On the day before treatment the back and flanks of each animal were clipped free of hair.
Using available information on the toxicity of the test item, a single group of animals was treated as follows:

Dose Level Specific Gravity Dose Volume Number of Rats
(mg/kg) (ml/kg) Male Female
2000 1.077 1.86 5 5


The calculated volume of test item, as received, was applied as evenly as possible to an area of shorn skin (approximately 10% of the total body surface area) using a graduated syringe. A piece of surgical gauze was placed over the treatment area and semi occluded with a piece of self adhesive bandage. The animals were caged individually for the 24 Hour exposure period. Shortly after dosing the dressings were examined to ensure that they were securely in place.
After the 24 Hour contact period the bandage was carefully removed and the treated skin and surrounding hair wiped with cotton wool moistened with distilled water to remove any residual test item. The animals were returned to group housing for the remainder of the study period.
The animals were observed for deaths or overt signs of toxicity ½, 1, 2 and 4 hours after dosing and subsequently once daily for fourteen days.
After removal of the dressings and subsequently once daily for fourteen days, the test sites were examined for evidence of primary irritation and scored according to the following scale from Draize J H (1977) "Dermal and Eye Toxicity Tests" In: Principles and Procedures for Evaluating the Toxicity of Household Substances, National Academy of Sciences, Washington DC p.31:

EVALUATION OF SKIN REACTIONS
Erythema and Eschar Formation Value

No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beef redness) to slight eschar formation (injuries in depth) 4

Oedema Formation

No oedema 0
Very slight oedema (barely perceptible) 1
Slight oedema (edges of area well-defined by definite raising) 2
Moderate oedema (raised approximately 1 millimetre) 3
Severe oedema (raised more than 1 millimetre and extending beyond the area of exposure) 4

Any other skin reactions, if present were also recorded.
Individual bodyweights were recorded prior to application of the test item on Day 0 and on Days 7 and 14.
At the end of the study the animals were killed by cervical dislocation. All animals were subjected to gross necropsy. This consisted of an external examination and opening of the abdominal and thoracic cavities. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.

Statistics:

No statistical analysis was performed.

Results and discussion

Preliminary study:

no pretest performed

Effect levels

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: 95% confidence limits not reported.

Mortality:

There were no deaths.

Clinical signs:

other: There were no signs of systemic toxicity.

Gross pathology:

No abnormalities were noted at necropsy.

Other findings:

Dermal Reactions: There were no signs of dermal irritation.

Any other information on results incl. tables

Evaluation of Data:

Data evaluations included the relationship, if any, between the exposure of the animal to the test item and the incidence and severity of all abnormalities including behavioural and clinical observations, gross lesions, bodyweight changes, mortality and any other toxicological effects.

Using the mortality data obtained, an estimate of the acute dermal median lethal dose (LD₅₀) of the test item was made.

The results were also interpreted according to Regulation (EC) No. 1272/2008 for classification and labelling of dangerous substances and the Globally Harmonised System of Classification and Labelling of Chemicals.

Table 1              Individual Clinical Observations and Mortality Data

Dose Level mg/kg	Animal Number and Sex	Effects Noted After Initiation of Exposure (Hours)				Effects Noted After Initiation of Exposure (Days)
		½	1	2	4	1	2	3	4	5	6	7	8	9	10	11	12	13	14
2000	1-0 Male	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-1 Male	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-2 Male	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-3 Male	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-4 Male	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	2-0 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	2-1 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	2-2 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	2-3 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	2-4 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

0 = No signs of systemic toxicity

Table 2              Individual Dermal Reactions - Males

Dose Level mg/kg	Animal Number and Sex	Observation	Effects Noted After Initiation of Exposure (Days)
			1	2	3	4	5	6	7	8	9	10	11	12	13	14
2000	1-0 Male	Erythema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Oedema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-1 Male	Erythema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Oedema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-2 Male	Erythema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Oedema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-3 Male	Erythema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Oedema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-4 Male	Erythema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Oedema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	0	0	0	0	0	0	0	0	0	0

0 = No reactions

Table 4              Individual Bodyweights and Weekly Bodyweight Changes

Dose Level mg/kg	Animal Number and Sex	Bodyweight (g) at Day			Bodyweight Change (g) During Week
		0	7	14	1	2
2000	1-0 Male	244	275	293	31	18
	1-1 Male	234	259	290	25	31
	1-2 Male	239	275	298	36	23
	1-3 Male	234	254	280	20	26
	1-4 Male	226	252	273	26	21
	2-0 Female	201	211	222	10	11
	2-1 Female	211	233	243	22	10
	2-2 Female	205	214	224	9	10
	2-3 Female	224	225	236	1	11
	2-4 Female	205	212	224	7	12

Table 5              Individual Necropsy Findings

Dose Level mg/kg	Animal Number and Sex	Time of Death	Macroscopic Observations
2000	1-0 Male	Killed Day 14	No abnormalities detected
	1-1 Male	Killed Day 14	No abnormalities detected
	1-2 Male	Killed Day 14	No abnormalities detected
	1-3 Male	Killed Day 14	No abnormalities detected
	1-4 Male	Killed Day 14	No abnormalities detected
	2-0 Female	Killed Day 14	No abnormalities detected
	2-1 Female	Killed Day 14	No abnormalities detected
	2-2 Female	Killed Day 14	No abnormalities detected
	2-3 Female	Killed Day 14	No abnormalities detected
	2-4 Female	Killed Day 14	No abnormalities detected

Applicant's summary and conclusion

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The acute dermal median lethal dose (LD50) of the test item in the Wistar strain rat was found to be greater than 2000 mg/kg bodyweight.

Executive summary:

The study was performed according to OECD Guidelines No. 402 and EU-Method B3. A group of ten animals (five males and five females) was given a single, 24 hour, semi‑occluded dermal application of the undiluted test item to intact skin at a dose level of 2000 mg/kg bodyweight. There were no deaths and no signs of systemic toxicity. All animals showed expected gains in bodyweight over the study period. No abnormalities were noted at necropsy. The acute dermal median lethal dose (LD50) of the test item in the Wistar strain rat was found to be greater than 2000 mg/kg body weight.