Ethyl acetoacetate

Endpoint summary

Description of key information

The test item EAA was tested in acute studies by oral and dermal application on rats. The acute oral study was performed before implementation of the corresponding

OECD- and GLP-guidelines in 1975, however it provides sufficient information to assess it as key study having Klimisch 2. Acute inhalation toxicity was assessed based on a read-across to the supporting substances Methylacetate and Ethylacetate. An additional Klimisch 3, non-guideline conform study with inhalative, intraperitoneal and oral application is available, but was not further assessed due to its poor quality and limited information).

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:

1975

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Deviations:

no

Principles of method if other than guideline:

The study was performed before GLP- and OECD-testing guidelines were available and in force.

A group of approximately 70 albino male and female rats, fasted for twenty-four hours were employed to establish an LD50 range for each product under test. Young adult rats which had not been used for previous test purposes were assigned to various dose levels at random. Both sexes were equally distributed. Body weight of the rats were 200-300 grams at the beginning of the study. Animals on the same dosage level were then placed in a common cage with free access to food and water. The animals were observed daily for a two week period.

No postmortem, or histopathology examinations were performed in this particular study.

GLP compliance:

no

Remarks:

GLP-guidelines not yet in force at date of the study

Test type:

standard acute method

Limit test:

no

Specific details on test material used for the study:

no data

Species:

rat

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

Fasted albino rats were used in this study.

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

The product under test was placed in a glass syringe and introduced through the esophagus into the stomach with a stainless steel catheter.

Doses:

7 dose groups (males & females) with dosages of:
- 0 mg/kg (control)
- 2000 mk/kg
- 4000 mg/kg
- 6400 mg/kg
- 8000 mg/kg
- 10000 mg/kg
- 12600 mg/kg
- 16000 mg/kg

No. of animals per sex per dose:

Five animals per sex and dose

Control animals:

yes

Details on study design:

no data

Statistics:

no data

Preliminary study:

No preliminary test performed.

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

12 300 mg/kg bw

Based on:

test mat.

95% CL:

10 300 - 14 800

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

10 800 mg/kg bw

Based on:

test mat.

95% CL:

9 300 - 12 500

Mortality:

No mortality was observed up to dose concentrations of 6400 mg/kg in males and up to 8000 mg/kg. Animals were found dead at 8000 mg/kg and above in males and at 10000 mg/kg and above in females. Details are given in the table below.

Clinical signs:

other: MALES: Males dosed at 2.0 g/kg and 4.0 g/kg exhibited moderate diarrhea for 2-3 days following intubation. Lethargy and moderate to severe diarrhea were noted at 6.4 g/kg and 8.0 g/kg. Rapid erratic respiration, lethargy, severe diarrhea, ruffled and unke

Gross pathology:

No postmortem, or histopathology examinations were performed in this particular study.

Other findings:

none

Mortality:

Dose Level (mg/kg)	No. of deaths (males)	Number of deaths (females)
2000	0	0
4000	0	0
6400	0	Not tested
8000	1 (day 2)	0
10000	1 (day 1)	2 (day 1 and 2)
12600	2 (day 1 and day 2)	4 (3 on day 1 and 1 on day 2)
16000	5 (day 1)	5 (day 1)

 

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The acute oral toxicity on Albino rats was determined to be 12300 mg/kg for males and 10800 mg/kg for females.

Executive summary:

A study was carried out equivalent or similar to EU Method B.1 and OECD Guideline 401 (Acute Oral Toxicity). 6 groups of 10 rats (5 male, 5 female) were treated by gavage with doses from 2000 up to 16000 mg/kg.Males and females dosed at 2.0 g/kg and 4.0 g/kg exhibited moderate diarrhea for 2-3 days following intubation. Lethargy and moderate to severe diarrhea were noted at 6.4 g/kg (only males tested) and 8.0 g/kg (both sexes). Rapid erratic respiration, lethargy, severe diarrhea, ruffled and unkempt coats were evident at the 10.0 g/kg and 12.6 g/kg dosage levels in both sexes. Death animals were observed at dose levels of 6400 mg/kg and above; for details refer to the above mentioned table. At 16000 mg/kg, the animals succumbed within 30 minutes after forced feeding. The LD50 was determined to be 12300 mg/kg for females and 10800 mg/kg for males.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

10 800 mg/kg bw

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Read-across from supporting substance: - Substance: Methyl acetate - CAS no.: 79-20-9 - EC no.: 201-185-2 Read-across justification: Ethyl acetoacetate is a well defined mono-constitutent substance of high purity. Based on the similar structure and properties, the substance Methyl acetate was selected as representative model structure for read-across. Compared to EAA, the selected structure show similar physical-chemical, toxicological and ecotoxicological properties. A data matrix covering all available endpoints was previously published in the EU-Risk assessment Report Vol. 34 of 2003. The available data on EAA and selected model constituent revealed similar properties. In general, the classification and labeling of the model constituent is comparable to that of Eethyl acetoacetate. Neither the model constituents nor MAA are considered PBT or vPvB substances. In summary, the read-across between MAA and the model constituent is justified. Result: Smyth et al screened industrial chemicals for acute toxicity values. For methylacetate the following values were determined: - LC0 = 49.2 mg/l/4h - LC50 = > 49.2 mg/l/4h

Qualifier:

no guideline followed

Principles of method if other than guideline:

Concentrated vapor inhalation consists of exposing 6 male or female albino rats to a following stream of vapor-loaded air. The vapor-air mixture is generated by passing 2.5L /minute of dried air at room temperature through a fritted glass disc immersed to a depth of at least one inch in approximately 50 ml of the test chemical contained in a gas-washing bottle. Inhalations are continued for time periods in a logarithmic series with a ratio of two extending from one-fourth to eight hours, until the inhalation period killing about half the number of rats within 14 days is defined. Inhalation of metered vapor concentrations by rats is conducted with flowing streams of vapor prepared by various styles of proportioning pumps. Inhalation periods are usually four hours' duration unless slight toxicity enforces an eight hour period. Concentration recorded are nominal and not analytically verified. They are in an essentially logarithmic series with a factor of two, and the Table records the concentration yielding fractional mortality among 6 rats within 14 days. Where no fractional mortality was observed, usually both the concentration yielding no mortality and that yielding complete mortality are indicated.

GLP compliance:

no

Test type:

standard acute method

Limit test:

no

Species:

rabbit

Strain:

other: Albino

Sex:

male/female

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Analytical verification of test atmosphere concentrations:

no

Duration of exposure:

4 h

Concentrations:

16,000 ppm and 32,000 ppm

No. of animals per sex per dose:

6 animals

Control animals:

no

Dose descriptor:

LC0

Effect level:

49.2 mg/L air

Exp. duration:

4 h

Dose descriptor:

LC50

Effect level:

> 49.2 mg/L air

Exp. duration:

4 h

49.2 mg/l < LC50< 98.4 mg/l

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Read-across to Methyl acetate:
The acute inhalative LC50 (4hr) of Methyl acetate on Albino rabbits was determined to be > 49.2 mg/L air.

Executive summary:

Read-across from supporting substance:

- Substance: Methyl acetate

- CAS no.: 79 -20 -9

- EC no.: 201 -185 -2

Smyth et al screened industrial chemicals for acute toxicity values. For methylacetate the following values were determined:

- LC0 = 49.2 mg/l/4h

- LC50 = > 49.2 mg/l/4h

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

49.2 mg/m³ air

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:

Between 01 February 2012 and 15 February 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 5350
- Expiration date of the lot/batch: 28-Nov-2013

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stable

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.

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 male
5 female

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 1.027 1.95 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.

Preliminary study:

no pretest performed

Key result

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
Individual dermal reactions are given in Table 2 and Table 3. (attachments 2 and 3)
Very slight erythema was noted at the test sites of two females two days after dosing. No other signs of dermal irritation were noted.

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 and the Globally Harmonised Classification System.

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 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	248	267	305	19	38
	1-1 Male	233	254	283	21	29
	1-2 Male	251	283	315	32	32
	1-3 Male	248	270	301	22	31
	1-4 Male	257	289	323	32	34
	2-0 Female	224	229	240	5	11
	2-1 Female	221	224	239	3	15
	2-2 Female	213	227	233	14	6
	2-3 Female	205	214	224	9	10
	2-4 Female	207	215	219	8	4

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

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. The test item did not meet the criteria for classification according to Regulation (EC) No. 1272/2008 or the Globally Harmonised Classification System.

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 abnormalitieswere 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Additional information

Key study: Acute toxicity – oral

A study was carried out equivalent or similar to EU Method B.1 and OECD Guideline 401 (Acute Oral Toxicity). 6 groups of 10 rats (5 male, 5 female) were treated by gavage with doses from 2000 up to 16000 mg/kg. Males and females dosed at 2.0 g/kg and 4.0 g/kg exhibited moderate diarrhea for 2-3 days following intubation. Lethargy and moderate to severe diarrhea were noted at 6.4 g/kg (only males tested) and 8.0 g/kg (both sexes). Rapid erratic respiration, lethargy, severe diarrhea, ruffled and unkempt coats were evident at the 10.0 g/kg and 12.6 g/kg dosage levels in both sexes. Death animals were observed at dose levels of 6400 mg/kg and above; for details refer to the above mentioned table. At 16000 mg/kg, the animals succumbed within 30 minutes after forced feeding. The LD50 was determined to be 12300 mg/kg for females and 10800 mg/kg for males.

Key study: Acute toxicity – inhalation

Read-across from the supporting substances Methylacetate (CAS no.: 79-20-9 / EC no.: 201-185-2) and Ethylacetate (CAS no.: 141-78-6 / EC no.: 205-500-4). Ethyl acetoacetate (EAA) is a well defined mono-constitutent substance of high purity. Based on the similar structure and properties, the substances Methyl- and Ethylacetate were selected as representative model structures for read-across. Compared to MAA, the selected structures show almost similar physical-chemical, toxicological and ecotoxicological properties. The vapor pressure of Ethyl acetoacetate is approx. two orders of magnitude lower than the vapor pressure of Ethyl acetate. A data matrix covering all available endpoints for Methylacetate was also published in the EU-Risk assessment Report Vol. 34 of 2003. The available data on EAA and selected model constituents revealed similar properties. For Methylacetate, Smyth et al (1962) reported a LC0 of 49.2 mg/l/4h and a LC50 of > 49.2 mg/l/4h on rabbits. For Ethylacetate, Smyth et al (1962)) reported a LC0 (4hr) of 29 mg/l; rats were able to withstand a vapour concentration of 8000ppm (~29mg/l) for 4 hours without mortality.

Key study: Acute toxicity – dermal

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 neither deaths nor clinical signs observed. There were no signs of dermal irritation, body weights remained unchanged and necropsy revealed no abnormalities. The LD50 was found to be > 2000 mg/kg body weight.

Justification for selection of acute toxicity – oral endpoint
non-GLP study following OECD-guideline; Klimisch 2

Justification for selection of acute toxicity – inhalation endpoint
Reliable data source; Klimisch 2

Justification for selection of acute toxicity – dermal endpoint
GLP study following OECD-guideline; Klimisch 1

Justification for classification or non-classification

Based on the data available the substance is not classified or labeled according to Directive 67/548/EEC (DSD) or Regulation 1272/2008/EC (CLP).