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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES:

The test substance was examined for mutagenic activity by assaying for reverse mutation to histidine prototrophy in the prokaryotic Salmonella typhimurium.

Five tester strains TA1535, TA1537, TA98, TA100 and TA102 were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with

phenobarbitone and betanaphthoflavone. Test substance solutions were prepared using DMSO.

Two independent experiments were performed, one using a plate incorporation method, the other using a pre-incubation method. The test substance was assayed at a maximum dose-level of 5000 µg/plate and four lower dose-levels,

separated by two-fold dilutions: 2500, 1250, 625 and 313 µg/plate.

The test substance did not induce two-fold increases of the number of revertant colonies in the plate-incorporation or pre-incubation assay, at any dose-level, in any tester strain, in the absence and presence of S9 metabolism.

CA:

The test compound did not induce a significant increase in the number of phases with aberrations at any preparation time and dose level of the test substance. No relevant cytotoxic effect of the compound was observed in the main experiments. Marked increases in the rate of chromosome aberrations were obtained with the positive control substances indicating the sensitivity of the assay.

In conclusion Acetoacetamide does not induce chromosome mutations V79 Chinese hamster cells, neither in the presence nor in the absence of a metabolic activation system, under the experimental conditions described.

HPRT

The test compound did not induce a significant increase in the number of mutant colonies or in the mutation frequency at any dose level of the test substance. No relevant cytotoxic effect of the compound was observed in the main experiments. Marked increases in mutation frequency were obtained with the positive control substances indicating the sensitivity of the assay.

In conclusion Acetoacetamid does not induce gene mutations in the HPRT-test with V79 Chinese hamster cells, either in the presence or in the absence of a metabolic activation system, under the experimental conditions described.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 November 1996 - 13 January 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and betanaphthoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
Toxicity test: 5000, 1580, 500, 158 and 50 µg/plate
Assay for reverse mutation: 5000, 2500, 1250, 625 and 313 µg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
cumene hydroperoxide
other: 2-aminoanthracene
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
Positive controls validity:
valid
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
Positive controls validity:
valid
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
Positive controls validity:
valid
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
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Slight toxicity in TA102 at the highest dose-level tested
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

The sterility of the S9 mix and the test substance solutions was confirmed by the absence of colonies on additional agar plates spread separately with these

solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control substances, indicating that the assay

system was functioning correctly.

Conclusions:
It can be concluded that the test item does not induce reverse mutation in Salmonella Typhimurium under the test conditions.
Executive summary:

The test substance was examined for mutagenic activity by assaying for reverse mutation to histidine prototrophy in the prokaryotic Salmonella typhimurium.

The five tester strains TA1535, TA1537, TA98, TA100 and TA102 were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone.

Test substance solutions were prepared using DMSO.

In the toxicity test, the test substance was assayed at a maximum dose-level of 5000 µg/plate and four lower dose-levels spaced at approximately half-log intervals. Slight toxicity, as indicated by thinning of the background lawn, was observed with TA102 tester strain at the highest dose-level tested. The same maximum dose-level was selected for the principal assay.

Two independent experiments were performed, one using a plate incorporation method, the other using a pre-incubation method. The test substance was assayed at a maximum dose-level of 5000 µg/plate and four lower dose-levels, separated by two-fold dilutions: 2500, 1250, 625 and 313 µg/plate.

The test substance did not induce two-fold increases of the number of revertant colonies in the plate-incorporation or pre-incubation assay, at any dose-level, in any tester strain, in the absence and presence of S9 metabolism.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 March 1989 - 24 May 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Purity: 98.2 and 98.4
Appearance: colourless crystals
Batch: 976 and 977
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9-mix
Test concentrations with justification for top dose:
101 μg/mL, 506 μg/mL, 1011 μg/mL - presence and absence of S9 mix
Vehicle / solvent:
aqua bid.
Untreated negative controls:
yes
Remarks:
untreated cells, untreated cells + S9-mix
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Details on test system and experimental conditions:
Two days old exonentially growing stock cultures which were over 50% confluent were trypsinised and a single cell suspension was prepared. The trypsin concentration was 0.25% in Ca-Mg-free salt solution.
After 24 hours the medium was replaced with medium containing 5% FCS and the test substance, both without S9-mix and with S9-mix.
After 4 hours this medium was replaced with normal medium after rinsing once with physiological saline solution.
Treatment was performed with three concentrations of the test substance.
4.5, 15.5 and 25.5 hours after the start of the treatment colcemide was added (0.04 μg/mL culture medium) to the cultures. 2.5 hours later (7h, 18h and 28h preparation) the cells were trypsinised. For hypotonic treatment, approximately 5 mL of 0.075 M potassium chloride solution at 37°C was quickly added and suspended. This suspension was the allowed to incubate for 10 minutes in a water bath at 37°C. Addition of 1.5 mL fixative and flow through with air.
After re-centrifuging for five miunutes at 1000 rpm, all but one drop of the supernatant was drawn off by pipette. The sediment was carefully covered layer composed of 2.5 mL fixative (methanol: glacial acetic acid 3 +1 ). After 20 min the fixation was removed with a pipette ans suspended in 2.5 mL fixative. After another 30 min, the mixture was centrifuged, after which the liquid was removed and fresh fixative added. The tubes were covered and kept for at least 12 hours (overnight) in a fridge at 4°C. 2-5 slidges were prepared from each flask, and stained.
Evaluation criteria:
The test substance is classified as mutagenic if it induces a significantly increased aberration rate as compared with the negative controls with one of the concentrations tested. The significance is obvious either by an enhancement of the rate clearly exceeding the control range or it is proven by adequate biometry (Binomial statistic with Fisher's exact test).
The test substance is classified as mutagenic if there is a reproducible concentration related increase in the aberration rate.
The test substance is classified as not mutagenic when it tests negatively both with and without metabolic activation.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control substances.
No relevant peproducible enhancement of metaphases with aberrations over the range of the negative control was found with any of the concentrations used, neighter with, nor without metabolic activation by S9 -mix.
Conclusions:
The results lead to the conclusion that the test compound is not mutagenic in the chromosome aberration test system in vitro with cells of the V79 Chinese hamster cell line.
Executive summary:

The chromosome aberration test was performed in year 1989 according to OECD Guideline 473 (In vitro Mammalian Chromosome Aberration Test) and GLP. The test substance Acetoacetamide was examined for mutagenic activity in V79 Chinese hamster cells. The induction of chromosome aberrations after in vitro treatment was investigated in the presence and absence of a fraction of liver homogenate for metabolic activation (S9 -mix).

A preliminary cytotoxicity experiment was performed in order to select appropriate dose levels for the mutagenicity study. The test substance produced no significant cytotoxic effect without metabolic activation from 5 μg/mL up to a concentration of 1011 μg/mL also observed with metabolic activation. For mutagenicity testing two independent cell cultures with and without metabolic activation (S9 -mix) up to the concentration of 1011 μg/mL were used. For main experiment dose levels of 101, 506 and 1011 μg/mL were used, in the absence and in the presence of S9 -mix metabolic activation.

The test compound did not induce a significant increase in the number of phases with aberrations at any preparation time and dose level of the test substance. No relevant cytotoxic effect of the compound was observed in the main experiments. Marked increases in the rate of chromosome aberrations were obtained with the positive control substances indicating the sensitivity of the assay.

In conclusion Acetoacetamide does not induce chromosome mutations V79 Chinese hamster cells, neither in the presence nor in the absence of a metabolic activation system, under the experimental conditions described.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 March 1989 - 10 August 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
Purity: 98.2 and 98.4
Appearance: colourless crystals
Batch: 976 and 977
Target gene:
HGPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9-mix
Test concentrations with justification for top dose:
with and without S9-mix:
100, 400, 700, 1011 µg/ml
Vehicle / solvent:
aqua bidest.
Untreated negative controls:
yes
Remarks:
Untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9,10-dimethylbenzanthracene
Details on test system and experimental conditions:
Two days old exonentially growing stock cultures which were over 50% confluent were trypsinised and a single cell suspension was prepared. The trypsin concentration was 0.25% in Ca-Mg-free salt solution.
After 24 hours the medium was replaced with medium containing 5% FCS and the test substance, both without S9-mix and with S9-mix.
After 4 hours this medium was replaced with normal medium after rinsing once with physiological saline solution.
Treatment was performed with three concentrations of the test substance.
4.5, 15.5 and 25.5 hours after the start of the treatment colcemide was added (0.04 μg/mL culture medium) to the cultures. 2.5 hours later (7h, 18h and 28h preparation) the cells were trypsinised. For hypotonic treatment, approximately 5 mL of 0.075 M potassium chloride solution at 37°C was quickly added and suspended. This suspension was the allowed to incubate for 10 minutes in a water bath at 37°C. Addition of 1.5 mL fixative and flow through with air.
After re-centrifuging for five miunutes at 1000 rpm, all but one drop of the supernatant was drawn off by pipette. The sediment was carefully covered layer composed of 2.5 mL fixative (methanol: glacial acetic acid 3 +1 ). After 20 min the fixation was removed with a pipette ans suspended in 2.5 mL fixative. After another 30 min, the mixture was centrifuged, after which the liquid was removed and fresh fixative added. The tubes were covered and kept for at least 12 hours (overnight) in a fridge at 4°C. 2-5 slidges were prepared from each flask, and stained.
Evaluation criteria:
The test substance is classified as mutagenic if it induces with one of the test substance concentrations reproducibly a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment.
The test substance is classified as mutagenic if there is a reproducible concentration-related increase in the mutation frequency. Such evaluation may be considered independently from the enhancement factor for induced mutants. However, in a case by case evaluation both decisions depend on the level of the corresponding negative control data.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
no significant cytotoxicity from 5 µg/ml up to a concentration of 1011 µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
The test compound did not induce a significant increase in the number of mutant colonies or in the mutation frequency at any dose level of the test substance. No relevant cytotoxic effect of the compound was observed in the main experiments. Marked increases in mutation frequency were obtained with the positive control substances indicating the sensitivity of the assay.
In conclusion Acetoacetamid does not induce gene mutations in the HPRT-test with V79 Chinese hamster cells, either in the presence or in the absence of a metabolic activation system, under the experimental conditions described.
Executive summary:

The test substance Acetoacetamid was examined for mutagenic activity in V79 Chinese hamster cells with two independent experiments. The induction of 6-thioguanine resistant mutants after in vitro treatment was investigated in the presence and absence of a fraction of liver homogenate for metabolic activation (S9-mix).

A preliminary cytotoxicity experiment was performed in order to select appropriate dose levels for the mutagenicity study. The test substance produced no significant cytotoxic effect from 5 µg/ml up to a concentration of 1011 µg/ml , which is the highest dose level tolerated for the test system. For mutagenicity testing two independent experiments with and without metabolic activation (S9·mix) up to a concentration of 1011 µg/ml were performed.

In the absence and in the presence of S9 metabolic activation dose levels of 100, 400, 700 and 1011 µg/ml were used for mutant selection in the main experiments.

The test compound did not induce a significant increase in the number of mutant colonies or in the mutation frequency at any dose level of the test substance. No relevant cytotoxic effect of the compound was observed in the main experiments. Marked increases in mutation frequency were obtained with the positive control substances indicating the sensitivity of the assay.

In conclusion Acetoacetamid does not induce gene mutations in the HPRT-test with V79 Chinese hamster cells, either in the presence or in the absence of a metabolic activation system, under the experimental conditions described.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

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