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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Alkylated Naphthalene was not mutagenic in the Ames test, a mammalian cell gene mutation test (MLA) and was did cause structural chromosome changes in a Chinese hamster Ovary study.
Link to relevant study records
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
Study period:
12 December 2006 to 06 February 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study without deficiencies
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 prepared in-house from the livers of male Sprague-Dawley rats
Test concentrations with justification for top dose:
Preliminary test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate.

Experiment 1: 50, 150, 500, 1500 and 5000 ug/plate
Experiment 2: 50, 150, 500, 1500 and 5000 ug/plate
Vehicle / solvent:
None
Details on test system and experimental conditions:
The test material was immiscible in both sterile distilled water and dimethyl sulphoxide at
50 mg/ml but was fully miscible in acetone at the same concentration in solubility checks
performed in-house. Acetone was therefore selected as the vehicle.
The test material was accurately weighed and approximate half-log dilutions prepared in acetone
by mixing on a vmtex mixer on the day of each experiment. Analysis for concentration,
homogeneity and stability of the test material formulations is not a requirement of the test
guidelines and was, therefore, not determined. Prior to use, the solvent was dried using molecular
sieves (sodium alumino-silicate) ie 2 tmn pellets with a nominal pore diameter of 4 x 10·4
microns.

S9 was prepared in-house on 12 November 2006 from the livers of male Sprague-Dawley rats
weighing - 250g. These had each orally received three consecutive daily doses of
phenobarbitone/~-naphthoflavone (80/l 00 mg per kg per day) prior to S9 preparation on Day 4.
Before use, each batch of S9 was assayed for its ability to metabolise appropriate indirect
mutagens used in the Ames Test. The S9 was stored at -196°C.

A 0.5 ml aliquot of S9-mix and 2 ml of molten, trace histidine or tryptophan supplemented, top
agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility
of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.
Top agar was prepared using 0.6% Difco Bacto agar (lot number 5279941 08/1 0) and 0.5%
sodium chloride with 5 ml of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution
added to each 100 ml of top agar. Vogel-Bonner Minimal agar plates were purchased from ILS
Limited (lot numbers 954961-02 05/11 & 961443-02 06/11).
Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in
revettant frequency over the dose range tested and/or a reproducible increase at one or more
concentrations in at least one bacterial strain with or without metabolic activation. Biological
relevance of the results will be considered first, statistical methods, as reconunended by the
UKEMS can also be used as an aid to evaluation, however, statistical significance will not be
the only determining factor for a positive response.

A test material will be considered non-mutagenic (negative) in the test system if the above criteria
are not met.
Although most experiments will give clear positive or negative results, in some instances the data
generated will prohibit a definitive judgement about the test material activity. Results of this type
will be reported as equivocal.

The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the
vehicle and untreated controls.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 10e9 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for
each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic
exposure and the integrity of the S9-mix. The historical control ranges for 2004 and 2005 were used.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contrunination.
Statistics:
Kirkland D J (Ed) (1989) Statistical Evaluation of Mutagenicity Test Data. UKEMS Subcommittee
on Guidelines for Mutagenicity Testing, Report - Part III, Cambridge University Press.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

Alkylated Naphthalene is not mutagenic in the Ames test.
Executive summary:

The substance was evaluated in the Bacterial Reverse Mutation Assay.

The method conforms to the guidelines for bacterial mutagenicity testing published by the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", and Method B13/14 of Commission Directive 2000/32/EC.

Methods. Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA' were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver 89 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 ug/plate in the first experiment. The experiment was repeated on a separate day using the same dose rm1ge as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. Results. The vehicle (acetone) control plates gave counts of revertant colonies within the nonnal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 ug/plate. A precipitate (oily in appearance) was observed at and above 1500 ug/plate, this did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Conclusion. Alkylated Naphthalene is not mutagenic in the Ames bacterial reverse mutation test.

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

Additional information

Additional information from genetic toxicity in vitro:

Based on the three GLP genetic toxicity tests conducted following current guidelines with Alkylated Naphthalene it can be concluded that the substance is not mutagenic or clastogenic.

Justification for classification or non-classification

Alkylated Naphthalene was not genotoxic in the required testing (three different in-vitro studies) and therefore is not classified for mutagenic effects according to CLP (Regulation EC No 1272/2008).