Pentan-2-one

Genetic toxicity: in vitro

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-Pentanone

IUPAC Name:

2-Pentanone

Constituent 2

Reference substance name:

Pentan-2-one

EC Number:

203-528-1

EC Name:

Pentan-2-one

Cas Number:

107-87-9

Molecular formula:

C5H10O

IUPAC Name:

pentan-2-one

Constituent 3

Reference substance name:

Methyl n-propyl Ketone

IUPAC Name:

Methyl n-propyl Ketone

Test material form:

other: Liquid

Details on test material:

Test Article: EC 98-0269, MPK, Lot No. 12-98
1. Physical Description: transparent colorless liquid
2. Date Received: 12/21/98

Method

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

Species / strain 1

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 2

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

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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'.

Applicant's summary and conclusion

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).