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Genetic toxicity in vitro

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Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted at a GLP compliant facility using OECD guidelines.
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535, and TA1537 as described by Ames et al. (1975) and the E Coli strain WP2uvrA as described by Green and Muriel (1976) and Brusick et al. (1980) containing the pKM101 plasmid.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TAl00, TA1535, and TA1537 as described by Ames et al. (1975). In addition to a mutation in the histidine operon, the tester strains contain two additional mutations which enhance their sensitivity to some mutagenic compounds. The rfa wall mutation results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide barrier that forms the surface of the bacterial cell wall. The resulting cell wall deficiency increases permeability to certain classes of chemicals such as those containing large ring systems (i.e. benzo(a)pyrene) that would otherwise be excluded by a normal intact cell wall. The second mutation, a deletion of the uvrB gene, results in a deficient DNA excision repair system which greatly enhances the sensitivity of these strains to some mutagens. Since the uvrB deletion extends through the bio gene, all of the tester strains containing this deletion require the vitamin biotin for growth.
Strains TA98 and TAl00 also contain the R-factor plasmid, pKMl0l, which further increases the sensitivity of these strains to some mutagens. The mechanism by which this plasmid increases sensitivity to mutagens has been suggested to be by modifying an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process.Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to
histidine independence (prototrophy) by frameshift mutagens. TA1535 is reverted by base substitution mutagens and TA100 is reverted by mutagens which cause both frameshifts and base
substitution mutations.
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Details on mammalian cell type (if applicable):
The tester strain used was the tryptophan auxotroph WP2uvr A as described by Green and Muriel (1976) and Brusick et al. (1980) containing the pKM101 plasmid. In addition to a mutation in the tryptophan operon, the tester strain contains a uvrA DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability since the uvr A repair system would normally act to
remove the damaged part of the DNA molecule and accurately repair it afterwards. Tester strain WP2uvrA(pKM101) also contains the R-factor plasmid, pKM101, which further increases the sensitivity of this strain to some mutagens. The mechanism by which this plasmid increases sensitivity to mutagens has been suggested to be by modifying an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process.
Tester strain WP2uvr A(pKM 101) is reverted from tryptophan dependence (auxotrophy) to tryptophan independence (prototrophy) by base substitution mutagens.
Metabolic activation:
with and without
Metabolic activation system:
b-naphthoflavone and phenobarbital
Test concentrations with justification for top dose:
100, 333, 1000, 3330, 5000 ug/plate with and without S9
Vehicle / solvent:
Water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
other: 2-Aminoanthracene, ICR-191
Details on test system and experimental conditions:
The assay was performed using tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA(pKM101) both in the presence and absence of S9 mix. Five doses of test article were tested along with the appropriate vehicle and positive controls. The doses of test article were selected based on the results of the dose range finding study. The results of the initial mutagenicity assay were confirmed in an independent experiment.

The tester strains were exposed to the test article via the plate incorporation methodology originally described by Ames et al. (1975) and Maron and Ames (1983). This methodology has been shown to detect a wide range of classes of chemical mutagens. In the plate incorporation methodology, the test article, the tester strain and the S9 mix (where appropriate) were combined in molten agar which was overlaid onto a minimal agar plate. Following incubation at 37 ± 2 C for 48 ± 8 hr, revertant colonies were counted. All doses of the test article, the vehicle controls, and the positive controls were plated in triplicate.

Each plate was labeled with a code which identified the test article, test phase, tester strain, activation condition, and dose. The S9 mix and dilutions of the test article were prepared activation condition, and dose immediately prior to their use.

When S9 mix was not required, 100 ul of tester strain and 100 ul of vehicle or test article dose were added to 2.5 ml of molten selective top agar (maintained at 45 ± 2 °C). When S9 mix was required, 500 ul of S9 mix, 100 ul of tester strain, and 100 ul of vehicle or test article dose were added to 2.0 ml of molten selective top agar. After the required components had been added, the mixture was vortexed and overlaid onto the surface of 25 ml of minimal bottom agar contained in a 15 x 100 mm petri dish. After the overlay had solidified, the plates were inverted and incubated for 48 ± 8 hr at 37 ± 2 C. Positive control articles were plated using a 50 ul plating aliquot.

Evaluation criteria:
Plates which were not evaluated immediately following the incubation period were held at 5 ± 3 C until such time that colony counting and bacterial background lawn evaluation could take place.

The condition of the bacterial background lawn was evaluated for evidence of cytotoxicity and test article precipitate. Evidence of cytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all plates at that dose on the data tables using the code system presented at the end of the Materials and Methods Section.

The number of revertant colonies per plate for the vehicle controls and all plates containing test article were counted manually. The number of revertant colonies per plate or the positive controls were counted by automated colony counter.
Statistics:
For all replicate platings, the mean revertants per plate and the standard deviation were calculated.
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and 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
Additional information on results:
The results of the dose rangefinding study were used to select the five doses tested in the mutagenicity assay. The doses tested were 5,000, 3,330, 1,000, 333, and 100 ug per plate in both the presence and absence of S9 mix. In the initial mutagenicity assay and in the confirmatory assay all data were acceptable and no positive increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix. All criteria for a valid study we:re met
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The results of the dose rangefinding study were used to select the five doses tested in the mutagenicity assay. The doses tested were 5,000, 3,330, 1,000, 333, and 100 ug per plate in both the presence and absence of S9 mix. In the initial mutagenicity assay and in the confirmatory assay, all data were acceptable and no positive increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix.
All criteria for a valid study were met
Executive summary:

This assay evaluated the test article and/or its metabolites for their ability to induce reverse mutations at the histidine locus in the genome of specific Salmonella typhimurium tester strains and at the tryptophan locus in an Escherichia coli tester strain both in the presence and absence of an exogenous metabolic activation system of mammalian microsomal enzymes derived from Aroclor™-induced rat liver (89).

The doses tested in the mutagenicity assay were selected based on the results of a dose rangefinding study using tester strains TA100 and WP2uvrA(pKM101) and ten doses of test article ranging from 5,000 to 6.67 ug per plate, one plate per dose, both in the presence and absence of S9 mix. The tester strains used in the mutagenicity assay were Salmonella typhimurium TA98, TA100,TA1535, and TA1537 tester strains and Escherichia coli tester strain WP2uvrA(pKM101). The assay was conducted with five doses of test article in both the presence and absence of S9 mix along with concurrent vehicle and positive controls using three plates per dose. The doses tested were 5,000, 3,330, 1,000, 333, and 100 ug per plate in both the presence and absence of S9 mix. The results of the initial mutagenicity assay were confirmed in an independent experiment. The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that under the conditions of this study, the test article did not cause a positive increase in the mean number of revertants per plate with any of the tester strains in either the presence or absence of microsomal enzymes prepared from Aroclor™-induced rat liver (S9).

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

Additional information

Additional information from genetic toxicity in vitro:

Ames Assay:

This assay evaluated the test article and/or its metabolites for their ability to induce reverse mutations at the histidine locus in the genome of specific Salmonella typhimurium tester strains and at the tryptophan locus in an Escherichia coli tester strain both in the presence and absence of an exogenous metabolic activation system of mammalian microsomal enzymes derived from Aroclor™-induced rat liver (89).

The doses tested in the mutagenicity assay were selected based on the results of a dose rangefinding study using tester strains TA100 and WP2uvrA(pKM101) and ten doses of test article ranging from 5,000 to 6.67 ug per plate, one plate per dose, both in the presence and absence of S9 mix. The tester strains used in the mutagenicity assay were Salmonella typhimurium TA98, TA100,TA1535, and TA1537 tester strains and Escherichia coli tester strain WP2uvrA(pKM101). The assay was conducted with five doses of test article in both the presence and absence of S9 mix along with concurrent vehicle and positive controls using three plates per dose. The doses tested were 5,000, 3,330, 1,000, 333, and 100 ug per plate in both the presence and absence of S9 mix. The results of the initial mutagenicity assay were confirmed in an independent experiment. The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that under the conditions of this study, the test article did not cause a positive increase in the mean number of revertants per plate with any of the tester strains in either the presence or absence of microsomal enzymes prepared from Aroclor™-induced rat liver (S9).

Chromosomal Aberration:

The objective of this in vitro assay was to evaluate the ability of EC 98-0269, MPK to induce chromosomal aberrations in Chinese hamster ovary (CHO) cells with and without metabolic activation. The test substance was dissolved in cell culture grade water at a concentration of 45.0 ug/ml for both the initial and confirmatory assays. The high dose achieved, 900 ug/ml, was >10 mM of the test substance. All dosing was achieved using a dosing volume of 2.0% (20.0 ug/ml) and the solvent control cultures were treated with 20.0 ug/ml of cell culture grade water. In the initial chromosomal aberrations assay, the treatment period was for 3.0 hours with and without metabolic activation and cultures were harvested 20.0 hours from the initiation of treatment. Concentrations of 6.14, 8.78, 12.5, 17.9, 25.6, 36.6, 52.4, 74.8, 107, 153, 218, 310, 442, 630, and 900 ug/ml were tested with and without metabolic activation. Cultures treated with concentrations of 310, 442, 630, and 900 ug/ml with and without metabolic activation were analyzed for chromosomal abeuations. No significant increase in cells with chromosomal aberrations was observed in the cultures analyzed. In a confirmatory chromosomal aberrations assay, the treatment period was for 17.8 hours without metabolic activation and 3.0 hours with metabolic activation, and cultures were harvested 20.2 hours from the initiation oftreatment. Concentrations of 71.6, 143, 286, 382, 508, 676, and 900 ug/ml were tested without metabolic activation, and concentrations of 286, 382, 508, 676, and 900 ug/ml were tested with metabolic activation. Cultures treated with concentrations of 382, 508, 676, and 900 ug/ml with and without metabolic activation were analyzed for chromosomal aberrtions. No significant increase in cells with chromosomal aberrations was observed in the cultures analyzed. MPK was considered negative for inducing chromosome aberrations in CHO cells with and without metabolic activation.

In Vitro Mammalian mutagenicity

The test was performed in two independent experiments in the absence and presence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).  The study procedures were based on the most recent OECD and EC guidelines. In the first experiment, Methyl Propyl Ketone was tested up to concentrations of 861 µg/ml (~0.01 M) in the absence and presence of 8 % (v/v) S9 -mix. The incubation time was 3 hours. In the second experiment, Methyl Propyl Ketone was again tested up to concentrations of 861 µg/ml, but in the absence and presence of 12% (v/v) S9-mix. The incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9-mix, respectively. No toxicity was observed at this dose level in the absence and presence of S9-mix. This is the highest concentration recommended in the guidelines. In the absence of S9-mix, Methyl Propyl Ketone did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time. In the presence of S9-mix, Methyl Propyl Ketone did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation. It is concluded that Methyl Propyl Ketone is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described.


Justification for selection of genetic toxicity endpoint
Note that only one study can be selected for this endpoint. All three of the studies conducted gave negative results.

Justification for classification or non-classification

As all studies were negative, no classification is required