Isopropyl acetoacetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was not mutagenic in bacteria strains in the absence or presence of metabolic activation (reference 7.6.1 -1).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996-10-22 to 1996-11-01

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983-05-26

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Version / remarks:

September 1985

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

not specified

Principles of method if other than guideline:

The study was conducted according to OECD 471 from 1983. According to that guideline version only 4 strains were required.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : rat liver
- method of preparation of S9 mix: Sufficient S9 fraction was thawed immediately at room temperature before each test. One volume of S9 fraction (protein concentration 49.9 g/L for both experiments) was mixed with 9 volumes of the S9 cofactor solution, which was kept on ice until used. The concentrations of the different compounds in the S9-mix were: 8 mM MgCI2, 33 mM KCI, 5 mM glucose-6-phosphate, 4 mM NADP, 100 mM phosphate buffer pH 7.4
- volume of S9 mix used in agar: 0.5 mL
- quality controls of S9: Sterility test was done on plates and for each batch of S9 an independent validation was performed with a minimum of two different mutagens, e.g. 2-aminoanthracene and dimethylbenzanthracene, to confirm metabolic activation by microsomal enzymes,

Test concentrations with justification for top dose:

4, 20, 100, 500, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene With metabolic activation for all strains

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
Precise toxicity testing was performed as follows using dose levels selected on the basis of toxicity results in the first test: 0.1 mL of the different concentrations of the test compound were thoroughly mixed with 0.1 mL of 10^6 dilution of the overnight culture of TA 100 (designated TA 100 D) and plated with histidine and biotin rich top agar (3 plates per dose). The solvent control was compared with the number of colonies per plate in the presence of the test compound. Results were determined as a ratio of these values (= surviving fraction).

Evaluation criteria:

A test compound was classified as mutagenic if it had either of the following effects:
a) it produced at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawns.
b) it induced a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawns.
If the test substance did not produce reproducible increases of at least 2 times the concurrent solvent controls, at any dose level with any bacterial strain, it was considered to show no evidence of mutagenic activity in this system.
The test results had to be reproducible.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Table 1: Experiment 1

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium
	TA98	TA100	TA1535	TA1537
	Results without S9
DMSO	18.7 ± 3.2	137.0 ± 21.1	17.0 ± 2.6	12.0 ± 2.0
untreated	20.3 ± 6.1	163.3 ± 2.5	19.7 ± 3.1	10.7 ± 1.2
4	21.7 ± 6.4	128.3 ± 9.7	19.3 ± 2.5	14.7 ± 4.5
20	17.7 ± 0.6	109.3 ± 20.8	18.0 ± 3.0	10.3 ± 4.5
100	17.0 ± 1.7	117.7 ± 12.9	20.0 ± 4.0	14.0 ± 5.2
500	19.7 ± 0.6	124.0 ± 34.9	19.7 ± 2.9	11.3 ± 2.1
2500	18.0 ± 2.6	103.7 ± 17.9	19.3 ± 2.1	10.3 ± 4.2
5000	18.7 ± 2.1	62.7 ± 21.5	17.0 ± 1.0	11.7 ± 1.5
2-NF	567.3 ± 95.1
SA (1)		782.7 ± 21.0	397.7 ± 24.4
9-AA (50)				169.7 ± 18.6
	Results with S9
DMSO	22.7 ± 3.1	162.7 ± 13.1	20.3 ± 2.5	11.0 ± 3.6
untreated	22.7 ± 1.5	146.3 ± 21.7	13.7 ± 1.5	15.7 ± 2.1
4	26.3 ± 8.4	137.0 ± 2.6	19.0 ± 4.6	11.0 ± 1.0
20	25.0 ± 4.6	141.0 ± 14.1	21.0 ± 2.0	11.7 ± 3.8
100	20.7 ± 3.8	137.0 ± 11.5	20.7 ± 1.4	11.0 ± 5.0
500	19.7 ± 3.2	122.7 ± 16.7	22.7 ± 1.5	9.0 ± 3.0
2500	22.7 ± 1.2	132.3 ± 10.3	19.7 ± 4.0	10.7 ± 2.1
5000	18.0 ± 1.0	116.7 ± 30.6	22.0 ± 1.0	10.3 ± 3.5
2-AA (0.5)	1870.0 ± 71.6	1971.0 ± 128.7
2-AA (1)			194.0 ± 14.7	331.0 ± 48.0

Table 2: Experiment 2

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium
	TA98	TA100	TA1535	TA1537
	Results without S9
DMSO	24.0 ± 6.9	180.3 ± 12.3	25.0 ± 5.3	9.0 ± 1.7
untreated	33.3 ± 6.0	174.0 ± 9.5	23.0 ± 4.6	9.3 ± 2.5
4	29.0 ± 1.0	153.3 ± 9.7	17.7 ± 2.9	8.0 ± 2.0
20	29.0 ± 0.0	176.3 ± 30.0	20.0 ± 3.5	7.3 ± 2.3
100	25.7 ± 3.2	153.0 ± 22.1	18.0 ± 6.1	8.3 ± 2.1
500	30.0 ± 7.5	144.7 ± 15.4	22.0 ± 1.7	8.7 ± 0.6
2500	28.0 ± 5.0	113.7 ± 8.7	15.3 ± 2.5	7.3 ± 0.6
5000	32.3 ± 1.5	113.3 ± 16.2	20.0 ± 6.2	9.0 ± 1.7
2-NF	514.3 ± 49.9
SA (1)		659.0 ± 26.2	365.7 ± 21.0
9-AA (50)				182.7 ± 33.7
	Results with S9
DMSO	23.3 ± 4.5	170.3 ± 29.2	24.7 ± 2.5	9.3 ± 0.6
untreated	40.3 ± 4.9	194.7 ± 13.2	22.0 ± 1.0	9.3 ± 1.5
4	29.0 ± 1.7	181.0 ± 9.5	22.7 ± 1.2	9.3 ± 0.6
20	29.7 ± 4.5	172.7 ± 22.5	26.0 ± 8.2	8.7 ± 0.6
100	31.3 ± 3.2	189.3 ± 13.2	23.7 ± 6.4	11.3 ± 5.1
500	36.0 ± 3.5	178.3 ± 11.5	26.0 ± 6.2	10.3 ± 3.1
2500	27.3 ± 2.5	161.3 ± 7.8	19.7 ± 2.9	7.3 ± 1.2
5000	27.3 ± 2.5	168.3 ± 12.9	16.3 ± 2.5	9.0 ± 1.7
2-AA (0.5)	2125.7 ± 75.0	2158.7 ± 26.3
2-AA (1)			194.3 ± 28.7	473.3 ± 10.2

Conclusions:

The test item was not mutagenic in bacteria strains in the absence or presence of metabolic activation.

Executive summary:

A bacterial reverse mutation assay according to OECD 471 was conducted to evaluate the mutagenic potential of the test item. Bacterial strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 were treated with 4, 20, 100, 500, 2500 and 5000 µg test item/plate in the absence and presence of metabolic activation with S9-mix in two independent experiments with triplicates for each strain. Furthermore, positive, solvent and untreated controls were conducted. The test item did not precipitate on the plates up to the highest investigated dose of 5000 µg/plate and proved to be not toxic to the bacterial strains. In an additional toxicity test with a dilution of tester strain TA 100 (designated TA 100 D), which was performed in parallel with the second mutation experiment, no toxicity was found. The test item did not cause a significant increase in the number of revertant colonies with any of the tester strains either in the absence or in the presence of S9-mix in either mutation test. No dose-dependent effect was obtained. All positive controls produced significant increases in the number of revertant colonies. Thus, the sensitivity of the assay and the efficacy of the exogenous metabolic activation system were demonstrated. Based on results obtained it was concluded that the test item was not mutagenic in bacteria strains in the absence or presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicity in vitro

A bacterial reverse mutation assay according to OECD 471 was conducted to evaluate the mutagenic potential of the test item. Bacterial strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 were treated with 4, 20, 100, 500, 2500 and 5000 µg test item/plate in the absence and presence of metabolic activation with S9-mix in two independent experiments with triplicates for each strain. Furthermore, positive, solvent and untreated controls were conducted. The test item did not precipitate on the plates up to the highest investigated dose of 5000 µg/plate and proved to be not toxic to the bacterial strains. In an additional toxicity test with a dilution of tester strain TA 100 (designated TA 100 D), which was performed in parallel with the second mutation experiment, no toxicity was found. The test item did not cause a significant increase in the number of revertant colonies with any of the tester strains either in the absence or in the presence of S9-mix in either mutation test. No dose-dependent effect was obtained. All positive controls produced significant increases in the number of revertant colonies. Thus, the sensitivity of the assay and the efficacy of the exogenous metabolic activation system were demonstrated. Based on results obtained it was concluded that the test item was not mutagenic in bacteria strains in the absence or presence of metabolic activation.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item does not require classification for genetic toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the fifteenth time in Regulation (EU) 2020/1182.