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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
Study period:
June-July 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD 471 protocol study under GLP without significant deviations

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)
Deviations:
yes
Remarks:
see below
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Principles of method if other than guideline:
5 strains were used, but not one of the strains having AT base pairs at the primary reversion site (such as TA 102 or E. coli WP2
strains).
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Contains more impurities than submission substance

Sponsor's identification : GBS-5
Batch number : GLS 30P (see CoA in the report attached)
Date received : 23 June 1994
Description : white powder
Storage conditions : room temperature over silica gel

Composition according to Certificate of Analysis
Glutamic acid - N,N -diacetic acid, tetrasodium salt 70.70%

Method

Target gene:
reverse mutation to histidine independency
Species / strain
Species / strain / cell type:
other: TA1535, TA100, TA1537, TA1538, TA98
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
liver homogenate S9 mix, Aroclor 1254-induced
Test concentrations with justification for top dose:
312.5, 625, 1250, 2500, 5000 µg/plate
Vehicle / solvent:
sterile distiiled water
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
for TA100 and TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
for TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-o-phenylenediamine (4NOPD)
Remarks:
for TA1538
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
for TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
in S9 series for TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
in S9 series for all other strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)


DURATION
- Preincubation period: 10 hours
- Exposure duration: 48 hours
- Expression time (cells in growth medium): ???????????????
- Selection time (if incubation with a selection agent): ??????????????
- Fixation time (start of exposure up to fixation or harvest of cells): ?????????????


SELECTION AGENT (mutation assays): ??????????
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):


NUMBER OF REPLICATIONS: ???????????


NUMBER OF CELLS EVALUATED: ???????????????


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: ????????????????

Evaluation criteria:
Presence of dose-related and statistically significant increase in mutation rate in one or more strains of bacteria in the presence and/or absence of S9 mix at sub-toxic dose levels.
Statistics:
Dunnett's method of linear regression.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
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 applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: none present

RANGE-FINDING/SCREENING STUDIES:
Preliminary toxicity test carried out in TA100 at same levels (312.5-5000 µg/plate) did not show toxicity.

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

The test substance was found to be non-mutagenic in the Ames test using strains TA1535, TA1537, TA1538, TA98 and TA100.
Although these strains may not detect certain oxidising mutagens or cross-linikg agents, it is not expected that the test substance
will do so. Therefore, it is concluded that there is no evidence of mutagenic potential of the test substance in this bacterial test
system.
Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with GBS-5 by 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 S9 in standard cofactors).

The dose range was determined in a preliminary toxicity assay and was 8 t o 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh chemical formulations. In this case the dose range o f GBS-5 was 312.5 t o 5000 µg/plate. The method used conforms with the OECD Guidelines for the Testing of Chemicals, Protocol No. 471 and also with Method B14 in Commission Directive 92/69/EEC.

The solvent (sterile distilled water) control plates gave counts of revertant colonies within the normal range.

All positive control chemicals produced marked increases in the number of revertant colonies, both with and without the metabolising system.

GBS-5 caused no visible reduction in the growth of the bacterial lawn at any of the dose levels employed in any of the strains of Salmonella tested. GBS-5 was, therefore, tested up to the maximum recommended dose of 5000 µg/plate.

No significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose of GBS-5, either with or without metabolic activation. GBS-5 was found to be non-mutagenic under the conditions of this test.