Registration Dossier

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
Study period:
1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Reactive Blue 13 Na/Li-Salt

Method

Target gene:
Salmonella typhimurium histidine (his) reversion system
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: his(+), uvrB(-), rfa(+), R factor(+)for TA 98 and TA 100; R factor(-)for TA 1535 and TA 1537
Species / strain / cell type:
E. coli, other: E.coli WP2P and WP2PuvrA
Additional strain / cell type characteristics:
other: uvrA mutation
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix (Rat liver enzymes)
Test concentrations with justification for top dose:
100 - 5000 µg/plate
Vehicle / solvent:
water
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
for all strains with S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 100, TA 1535 without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Acridine Mutagen ICR191
Remarks:
TA 1537 without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Daunomycin HCl
Remarks:
TA 98 without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
E. coli WP2P without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
E. coli WP2P uvrA· without S9
Details on test system and experimental conditions:
A solution of the test compound (as supplied) was prepared in sterile (filtered: Milli-Q/RO) water to yield a w/v concentration of 50.00mg/ml.
This was then diluted with water to give additional solutions of 25mg/ml, 10mg/ml, 5.0mg/ml, 2.0mg/ml and 1.0mg/ml. Fresh stock solutions and dilutions were prepared as necessary for each experiment.
Water was also included as the negative (solvent) control.

The test substance was initially assayed using the standard plate incorporation protocol over a dose range of 5000 - 100µg per plate using all six strains, both in the presence and absence of a liver S9-mix prepared from Phenobarbital/ p-Naphthaflavone -induced Alderley Park (Alpk:APfSD). The compound was subsequently re-tested in all six strains over this same dose range: the +S9 phase of this repeat assay was conducted using the Pre-Incubation protocol. In light of the variable results observed with strain TA1535 (+S9) in these two experiments, the compound was re-tested in this strain (+S9 only) over the same dose range, using the plate incorporation protocol. The incubation period for each experiment was 3 days (of 37°C).
For each experiment, positive control compounds were tested to validate the bacterial strains and to confirm the activity of each batch of S9-mix used.
Evaluation criteria:
Test data from individual experiments are considered valid if:
a) the concurrent solvent control data are acceptable;
b) the positive control data show unequivocal positive responses;
c) at least the lowest test compound dose shows no evidence of toxicity, and at least three test doses show no significant toxicity (ie significant loss of background growth and/or reductions in colony numbers).
Failure of one or more tester strain/59 combinations does not invalidate the data for the remainder of a concurrent experiment.

A positive response in a (valid) individual experiment is achieved when one or both of the following criteria are met:
a) a statistically significant dose-related increase in the mean number of revertant colonies is obtained;
b) a two-fold or greater increase in the mean number of revertant colonies (over that observed for the concurrent solvent control plates) which is
statistically significant, is observed at at least one dose level.

A negative result in a (valid) individual experiment is achieved when:
a) There is no statistically significant dose-related increase in the mean number of revertant colonies per plate observed for the test compound; and
b) in the absence of any such dose response, no increase in colony numbers is observed (at any test dose) which exceeds 2x the concurrent solvent control.

For a positive response in an individual experiment to be considered indicative of an unequivocal positive, ie mutagenic, result for that strain/S9 combination, then the observed effect(s) must be consistently reproducible.
Statistics:
All derived calculations (ie mean colony count/plate; standard deviation, etc) shown in the results tables were carried out by computer.
Counts from contaminated plates are not included in these calculations.

An assessment of statistical significance was carried out using a onetailed Student's t-test (Ehrenberg 1984). The corresponding probability for each dose level was derived by computer using the appropriate degrees of freedom. Values of p<0.01 are treated as significant, with values of 0.01<=p<0.05 being indicative of a possible effect.

Results and discussion

Test resultsopen allclose all
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
Species / strain:
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
Species / strain:
E. coli WP2 uvr A
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:
IIn at least two separate assays, the compound did not induce any significant, reproducible increases in the observed number of revertant colonies in strains TA1535, TA1537, TA98, TA100, WP2P and WP2PuvrA either in the presence or absence of an auxiliary metabolising system (S9). Although a small increase in colony numbers was observed in the first plate incorporation experiment with strain TA1535 (+S9), this was at a single dose level (2500 µg) and was not reproducible in a further experiment. The isolated increase in strain TA1535 (+S9) in the pre-incubation assay is not dose-related and is clearly due to a single plate.

Applicant's summary and conclusion

Conclusions:
The test substance gave a negative, ie non-mutagenic response in S.typhimurium strains TA1535, TA1537, TA98 and TA100 and in E.coli strains WP2P and WP2PuvrA in both the presence and absence of S9.
Executive summary:

The test substance has been evaluated in a bacterial mutagenicity assay (Gatehouse et al,1990: based an Maron and Ames (1983)) using four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and two strains of Escherichia coli (WP2P and WP2PuvrA), following protocols complying with OECD Guideline Numbers 471 and 472 (OECD, 1983a and 1983b) and with the United Kingdom Department of Health Guidelines (DoH, 1989).

In at least two separate experiments, the compound did not induce any reproducible, significant increases in the observed numbers of revertant colonies in any of the tester strains used, either in the presence or absence of an auxiliary metabolising system (S9).In each experiment, the positive controls responded as expected indicating that the assay was performing satisfactorily.

Under the conditions of this assay, the test substance gave a negative, ie non-mutagenic response in S.typhimurium strains TA1535, TA1537, TA98 and TA100 and in E.coli strains WP2P and WP2PuvrA in both the presence and absence of S9.