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Toxicological information

Genetic toxicity: in vitro

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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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES are commonly accepted as trustworthy and useful reference books

Data source

Reference
Reference Type:
review article or handbook
Title:
Unnamed
Year:
1989

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only 4 strains used instead of the 5 required by the current guideline
Principles of method if other than guideline:
Preincubation assay as described by Haworth et al (1983): Environ Mutagen 5[Suppl 1], 3-142
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
histidine locus
Species / strain
Species / strain / cell type:
other: Salmonella typhimurium TA 98, TA100, TA1535, TA1537
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-liver fractions of male Sprague-Dawley rats and male Syrian hamsters induced with Ariclor 125
Test concentrations with justification for top dose:
0, 100, 333, 1000, 3333, 3000, and 6666 µg/plate
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
no
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylene diamine, 2-aminoanthracene
Details on test system and experimental conditions:
Pre-test: All chemicals were initially tested with strain TA100 in the presence and the absence of the metabolic activation systems, over a wide dose range with an upper limit of 10 mg/plate, or less when solubility problems were encountered.

To each of 13 x 100-mm test tubes maintained at 37°C were added in the following order: 0.5 ml of S-9 mix or 0.1 M P04 buffer (pH 7.4), 0.05 ml of the overnight culture, and 0.05 ml of solvent or chemical dilution. The mixture was mixed and allowed to incubate without shaking at 37°C for 20 min, at which time 2.5 ml of molten (45°C) top agar supplemented with 0.5 mM L-histidine and 0.5 mM D-biotin were added. The contents of the tubes were mixed and poured onto 25 ml of minimal glucose bottom agar [Vogel and Bonner, 1956] in 15 x 100-mm plastic petri dishes (Falcon Muta-Assay, 1028). When the top agar had solidified, the plates were inverted and incubated at 37°C for 48 hr.
Concurrent solvent and positive controls were tested with and without the metabolic activation systems. At least five dose levels of the chemicals were tested,
with three plates per dose level. All assays were repeated (as described above) no less than 1 wk after completion of the initial test.
Nontoxic chemicals were tested in the initial experiment up to the 10 mg/plate dose level, or to a level determined by their solubility. Toxic chemicals were tested up to a high dose which exhibited some degree of toxicity. As a rule, at least one toxic dose was incorporated into the first mutagenicity test; the repeat test(s) occasionally had the doses adjusted so that an apparent toxic dose was not reached.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity was evidenced by one or more of the following phenomena: appearance of his(neg) pinpoint colonies, reduced numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn.
Evaluation criteria:
positive response: dose-related reproducible increase in the number of revertants over background even if the increase was less than twofold
questionable response: absense of a clear-cut dose-reated increase of revertants or when the response was not reproducible
negative response: when no increase in the number of revertants was elicited by the chemical
Statistics:
no data

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Benzyl alcohol was not mutagenic when tested by the preincubation protocol in the presence or absence of exogenous metabolic activation in Salmonella typhimurium strains TA98, TA100, TA1535, or TA1537.
Executive summary:

A bacterial reverse mutation assay (Ames) according to the protocol of Haworth (1983) was conducted using the four Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100 with and without a metobolic activating system. The doses were based on a preliminary dose-setting experiment and ranged between 100 and 6666 µg/plate. Benzyl alcohol was not mutagenic when tested by the preincubation protocol in the presence or absence of exogenous metabolic activation in Salmonella typhimurium strains TA98, TA100, TA1535, or TA1537.