2-[(2-methyl-1-oxoallyl)oxy]ethyl acetoacetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

AMES STUDY: ------------ A study according to OECD Guideline 471 (Bacterial Reverse Mutation Assay) was carried in year 1990. Tested up to 5000 ug/plate without and with metabolic activation the test substance induced no statistically significant dose-related increase in the numbers of revertant (His+) colonies in each of the five tester strains (TA 1535; TA 1537; TA 1538; TA 98 and TA 100). There was no toxicity to the bacteria in any of the experiments performed. There was no precipitation of the test substance. It was concluded that the test item was not mutagenic to Salmonella typhimurium when tested in dimethylsulphoxide up to the predetermined maximum limit of 5000 µg per plate. CHROMOSOME ABERRATION: -------------------------- A chromosome aberration test was performed in year 2011 according to OECD Guideline 473 (In vitro Mammalian Chromosome Aberration Test) and GLP. It was concluded that LZ649 has shown evidence of causing an increase in the frequency of structural chromosome aberrations in this In-Vitro test, in the absence of S9 mix following continuous treatment only, in this In-Vitro cytogenetic test system under the experimental conditions described. MOUSE LYMPHOMA ASSAY: ------------------------ A mouse lymphoma assay was performed in year 2011 according to OECD Guideline 476 (In vitro Mammalian Cell Gene Mutation Test). It was concluded that LZ649 demonstrated mutagenic potential in this in vitro cell mutation assay, in the absence of S9 mix following 24 hour treatment only, under the experimental conditions described. MICRONUCLEUS ASSAY IN VIVO: ---------------------------- The in vivo study was peformed in year 2016 according to OECD 474 and GLP. The test substance does not produce relevant increases of the numbers of micronuclei in polychromatic erythrocytes after in vivo treatment of mice of either sex of the test strain at doses of 500, 1000 and 2000 mg/kg b.w. Therefore, the test item is considered to be non-mutagenic in this in vivo test.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 January 2016 - 3 March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline study and performed according to GLP requirements.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Envigo CRS GmbH

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

Number of animals for
the pre-test: 2 males and 2 females for each pre-test
Number of animals for
the main study: 42 males for the main study
9 additional males for determination of bioavailability
Age (beginning of treatment): 6 – 10 weeks
Body weight: between 32.1 g and 40.0 g
Acclimation: At least 5 days prior to the start of dosing under test conditions after health examination.
Only animals without any visible signs of illness were used for the study.

Route of administration:

oral: gavage

Vehicle:

70 % Polyethylene glycol

Details on exposure:

Application volume: 10 mL/kg b.w.

Duration of treatment / exposure:

The animals of all dose groups, except the positive control group, were examined for acute toxic symptoms at intervals of around 0-1 h, 2-4 h, 5-6 h, 24 h, and 48 h after administration of the test item. Sampling of the bone marrow was done 24 and 48 h after treatment, respectively.

Frequency of treatment:

1

Post exposure period:

Pre-Experiment on Toxicity: 0-1 h, 2-4 h, 5-6 h, 24 h, and 48 h after administration of the test item.
Main experiment: 0-1 h, 2-4 h, 5-6 h, 24 h, and 48 h after administration of the test item.


24h + 48h

Remarks:

Doses / Concentrations:
500 mg/kg b.w.
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1000 mg/kg b.w.
Basis:
actual ingested

Remarks:

Doses / Concentrations:
2000 mg/kg b.w.
Basis:
actual ingested

Remarks:

Doses / Concentrations:
Positive control 40 mg/kg b.w. Cyclophosphamide
Basis:
actual ingested

No. of animals per sex per dose:

Number of animals for the pre-test: 2 males and 2 females for each pre-test
Number of animals for the main study: 42 males for the main study
Main study: 7 males per test group
24 h preparation interval: 500, 1000, and 2000 mg/kg b.w.
48 h preparation interval: 2000 mg/kg b.w.

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide 40 mg/kg b.w.

Tissues and cell types examined:

Tissue: bone marrow
Cell type: polychromatic erythrocytes (PCE)

Details of tissue and slide preparation:

Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives.
4000 polychromatic erythrocytes (PCE) per animal were analysed for micronuclei.

Evaluation criteria:

To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per total erythrocytes. The analysis was performed with coded slides.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test using the validated statistical program RScript Wilcoxon_2.Rnw.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

clinical signs

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

As estimated by a pre-experiment 2000 mg LZ 649 per kg b.w. (the maximum guideline-recommended dose) was suitable as highest treatment dose.Clinical symptoms in the main experiment included ruffled fur, reduced spontaneous activity and eyelid closure in some of the animals treated with the high dose of test item. The animals treated with the mid and low dose did not exhibit any clinical symptoms.The observed systemic toxicity suggests a systemic distribution of the test item. Thus, bioavailability of the test item under the tested conditions is assumed. This was additionally confirmed by analytical detection of the test item in plasma (Study Number 41502651).
The mean number of polychromatic erythrocytes was not substantially decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that the test item did not have any cytotoxic properties in the bone marrow.
In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with the test item were below or near to the value of the vehicle control group.

	hours post-treatment (males)
Clinical symptoms	1	2-4	6	24	48
High dose: 2000 mg/kg b.w. (14 males at 1 to 24 h; 7 males at 48 h)
reduction of spontaneous activity	1	2	0	1	0
eyelid closure	0	2	0	0	0
ruffled fur	1	2	1	2	0

The animals treated with the vehicle control (PEG 400), or with the low and mid dose of the test item did not express any clinical symptoms.

Summary of Micronucleus Test Results:

Test Group	Dose mg/kg b.w.	Sampling time	Mean MN/4000 PCE	SD MN/4000 PCE	Range		Ratio PCE / total Ery	% ratio Vehicle
					min	max
Vehicle	0	24	4.1	2.6	1	9	0.572	100.00
Dose 1	500	24	4.7	2.6	2	8	0.577	100.87
Dose 2	1000	24	3.7	1.9	1	5	0.591	103.32
Dose 3	2000	24	2.6	1.6	0	4	0.621	108.57
Positive	40	24	90.4	33.8	51	144	0.654	114.34
Dose 3	2000	48	4.0	2.2	2	8	0.546	95.45

MN= micronuclei

PCE= polychromatic erythrocytes

Conclusions:

Interpretation of results (migrated information): negative
The test substance does not produce relevant increases of the numbers of micronuclei in polychromatic erythrocytes after in vivo treatment of mice of either sex of the test strain at doses of 500, 1000 and 2000 mg/kg b.w. Therefore, the test item is considered to be non-mutagenic in this in vivo micronucleus assay.

Executive summary:

The in vivo study was peformed to investigate the potential of the tests item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The study was performed in year 2016 according to OECD Guideline 474 and GLP.

The observed systemic toxicity suggests a systemic distribution of the test item. Thus, bioavailability of the test item under the tested conditions is assumed. This was additionally confirmed by analytical detection of the test item in plasma (supporting phase report 41502651).

After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test item did not exert any cytotoxic effects in the bone marrow.

In conclusion, in can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Therefore, the test item is considered to be non-mutagenic in this in vivo micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

An Ames test was performed. Tested up to 5000 ug/plate without and with metabolic activation the test substance induced no statistically significant dose-related increase in the numbers of revertant (His+) colonies in each of the five tester strains (TA 1535; TA 1537; TA 1538; TA 98 and TA 100). LZ649 was not mutagenic to Salmonella typhimurium when tested in dimethylsulphoxide up to the predetermined maximum limit of 5000 µg per plate.

The test item has shown evidence of causing an increase in the frequency of structural chromosome aberrations in the in-vitro test, in the absence of S9 mix following continuous treatment only, in this in-vitro cytogenetic test system under the experimental conditions described.

Furthermore, LZ649 demonstrated mutagenic potential in the in-vitro cell mutation assay (mouse lymphoma), in the absence of S9 mix following 24 hour treatment only, under the experimental conditions described.

Therefore, a in-vivo mutagenicity study shall be considered in case of a positive result in any of the genotoxicity studies.

The in vivo study was peformed to investigate the potential of the tests item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The study was performed in year 2016 according to OECD Guideline 474 and GLP.

The observed systemic toxicity suggests a systemic distribution of the test item. Thus, bioavailability of the test item under the tested conditions is assumed. This was additionally confirmed by analytical detection of the test item in plasma (supporting phase report 41502651).

After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test item did not exert any cytotoxic effects in the bone marrow.

In conclusion, in can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Therefore, the test item is considered to be non-mutagenic in this in vivo micronucleus assay.

Justification for selection of genetic toxicity endpoint
GLP compliant studies conducted in accordance with international guidelines.

Justification for classification or non-classification

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