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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:
other: GLP study performed in accordance with the OECD Guideline (471: Bacterial Reverse Mutation Assay). Cytotoxicity and solubility of the substance in the final treatment mixture were not determined.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Details on test material:
Description: Complex reaction product of an aminate base-carbonate and fatty acids
Chemical name: HiTEC 7134 Performance additive
Storage: in the dark at room temperature
Physical state: viscous liquid

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from phenobarbital/b-naphthoflavone- induced Sprague-Dawley rats
Test concentrations with justification for top dose:
100, 200, 500, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: Acridine mutagen, benzo[alpha]pyrene, 2-aminoanthracene, daunomycin HCl, N-ethyl-N'-nitro-N-nitrosoguanidine, mitomycin C, sodium azide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) AND preincubation

Overnight cultures (10-12 h) of each bacterial strain were seperated

PLATE INCORPORATION METHOD
0.5ml S9-mix (or S9 buffer) with 0.1ml of the appropriate concentration of test substance preparation added to each aliquot with one strain. Thereafter, 2 ml appropriately prepared top agar was added to each aliquot; this mixture was poured onto the surface of a prepared pre-labelled Vogel Bonner plate (9cm diameter vented Petri-dish prepared with 25ml Vogcl Bonner minimal medium and containing 1.5% w/v agar and 2% w/v glucose and allowed to gel. Plates were incubated inverted at 37 C for 3 days in the dark.

Following incubation the plates were examined and counted by an automated colony counter. Contaminated plates were recorded as such without counting.


PREINCUBATION METHOD
The procedure was as for the plate-incorporation protocol described above, except that
a) each compound/solvent dose was added in 0.02ml volumes, with the total volume made up to 0.1ml with phosphate buffered saline;
b)before adding the top agar, each compound/strain group of bijoux were placed on an orbital shaker (at approximately 140rpm) for 60 minutes (at 37°C).



Evaluation criteria:
The results were considered valid only if:
i) the solvent control data were acceptable, and
ii) the positive control data show unequivocal positive responses

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 one or more concentrations.
For a positive response in an individual experiment to be considered indicative of mutagenicity, the effect must be consistently reproducible.

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 substance; and
b) in the absence of any such dose response, no increase in colony numbers is observed (at any test concentration) which exceeds 2x the concurrent solvent control.
Statistics:
One sided t-test, 0.01≤p0.05, p<0.01

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:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli, other: WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Precipitation: the test substance precipitated at the two highest doses used


Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Experimental results can be found in the attached document below.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative compound precipitated at 2500µg/plate and above

HiTEC 7134 did exert a mutagenic effect to two tester strains under the conditions of this test. However, this effect was not reproducible. All the other results were negative. HiTEC 7134 can be considered as a non-mutagen in the Ames test, under these conditions.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA1535, TA1537, TA98, and TA100 ofS. Typhimurium,as well as WP2 pKM101 and WP2 uvr A pKM 101 ofE. coli  were exposed to the test substance HiTEC 7134 at concentrations of 100, 200, 500, 1000, 2500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation (S9 mix). Toxicity and solubility of the test material were not evaluated. The mutagenic potential of HiTEC 7134 was assessed in all six strains using the standard plate incorporation protocol. The test substance was subsequently re-tested in all strains; in this second assay the +S9-mix phase was performed with the use of the pre-incubation protocol. The results revealed an equivocal positive response in one of the assays without metabolic activation for the strains TA1535 and TA1537. A 2Xfold significant increase in the number of revertants was observed in some concentration levels, when tested without the S9 mixture; still, this increase was not dose-dependent, probably due to exerted cytotoxicity of the substance at higher concentration levels. In view of these results, the experiment was repeated for a 3rdtime for the strains TA1535 and TA1537. A positive response was not reproducible. The results were negative for all the other tester strains, both with and without metabolic activation.It can be concluded that, under the conditions of this test, HiTEC 7134 gave a negative, i.e. non-mutagenic response in all strains used, in both the presence and absence of metabolic activation.

This test was performed in accordance with the OECD Guideline 471.