2-Oxetanone, 3-C14-16-alkyl 4-C15-17-alkylidene derivs.

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

7 July 1998 - 13 July 1998.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed accordig to internationally accepted guidelines and under GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: The method described was also designed to comply with ICH (1996 & 1997), and followed the recommendations of the United Kingdom Environmental Mutagen Society (Gatehouse er al 1990).

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: US EPA 40 CFR Part 799 (1997) Toxic Substances Control Act Test Guidelines - Subsection 799.95 10, TSCA bacterial reverse mutation test. Federal Register, Vol . 62, No. 158.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

849705-80-2

Cas Number:

849705-80-2

IUPAC Name:

849705-80-2

Details on test material:

Indentity: P-2290
Chemical name: 2-Oxetanone, 3-(C14-16 and C16-unsatd. branched and linear alkyl) 4-(C15-17 and C17-unsatd.branched and linear alkylidene) derivs
Intended use: Paper Chemicals
Appearance: Waxy liquid
Storage conditions: 4°C in the dark
Batch number: 290/97
Purity: 88.9%

Method

Target gene:

Histidine operon

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: See "additional information on materials...."

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Additional strain / cell type characteristics:

other: See "additional information on materials...."

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Test 1: 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Test 2: 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Prior to commencing testing, the solubility of the test substance was assessed at 50 mg/ml in dirnethyl
sulphoxide and ethanol in which it was insoluble, and in acetone, in which it dissolved. Water was not assessed as the test substance was hown to be insoluble in the solvent. Therefore acetone was used as the solvent for this study. The solubility of the test substance at 200 mg/ml in acetone was also checked as it was likely that it would be dosed at this concentration if the pre-incubation assay was required.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: See "additional information on materials...."

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation), 1st test = standard test, 2nd test = pre-incubation test.

DURATION
- Preincubation period: 1st test: none, 2nd test: 30 minutes
- Exposure duration: 72 hours

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: triplicate

NUMBER OF CELLS EVALUATED: all colonies were counted

DETERMINATION OF CYTOTOXICITY
- Method: Any toxic effects of the test substance would be detected by a substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn.

OTHER:
Each batch of frozen strain was tested, where applicable, for cell membrane permeability (rfa mutation), sensitivity to UV light and the pKM101
plasmid which confers resistance to ampicillin. The responses of the strains to a series of diagnostic mutagens were also assessed.

Evaluation criteria:

For a test to be considered valid the mean of the solvent control revertant colony numbers for each strain should be in the range stated in the appropriate Standard Operating Procedure. These ranges are based on the laboratory's historical control values. Also, the positive control compounds must cause at least a doubling of mean revertant colony numbers over the solvent control. The mean number of revertant colonies for all treatment groups is compared with those obtained for the solvent control groups. The mutagenic activity of a test substance is assessed by applying the following criteria:
- If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix, it is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
- If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, in either mutation test, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
- If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response given in the paragraphs above, additional testing may be performed in order to resolve the issue of the test substance's mutagenic activity in this test system. Modifications to the experimental method will usually be considered, such as the use of a narrower dose range and different levels of S9 in the mix. Should an increase in revertant colony numbers then be observed whch satisfies the first paragraph the substance is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.

Statistics:

If no clear "positive" response can be obtained, the test data may be subjected to analysis to
determine the statistical significance of any observed increases in revertant colony numbers. The
statistical procedures used will be those described by Mahon et a1 (1989) and will usually be
analysis of variance followed by Dunnett's test.

Results and discussion

Test results

Test results 1

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

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:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: not water soluble
- Precipitation: none
- Other confounding effects: no data


RANGE-FINDING/SCREENING STUDIES:
Not performed

COMPARISON WITH HISTORICAL CONTROL DATA:
The mean revertant colony counts for the solvent controls were within the historical range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
None

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

It is concluded that, when tested in acetone, P-2290 shows no evidence of mutagenic activity in this bacterial system.

Executive summary:

According to internationally accepted guidelines and under GLP this in vitro study assesses of the mutagenic potential of P-2290, histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA1535, TA1537, TA98 and TA100) and a tryptophan dependent mutant of Escherichia coli (strain CM891) were exposed to the test substance diluted in acetone, which was also used as a negative control. Two independent mutation tests were performed in the presence and absence of liver preparations from Aroclor 1254-induced rats (S9 mix). The first was a standard plate incorporation assay, the second involved a pre-incubation stage. Dose levels of up to 5000 µg/plate were tested in the mutation tests. This is the standard limit dose recommended in the regulatory guidelines this assay follows. Other dose levels used were a series of ca half-loglo dilutions of the highest concentration. No signs of toxicity were observed towards the tester strains in either mutation test. No evidence of mutagenic activity was seen at any dose level of P-2290 in either mutation test. The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It is concluded that, when tested in acetone, P-2290 shows no evidence of mutagenic activity in this bacterial system.