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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 23, 1988 - Aug 19, 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with minor deviations from standard test guidelines which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
The temperature was recorded to be outside the range of 37.0 ± 1.0 C specified in the protocol for approx.75 min in the second mutation experiment (with a maximum of 38.4 C). The study integrity was not adversely affected by the deviation.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): OS81067
- Physical state: amber liquid
- Storage condition of test material: At room temperature

Method

Target gene:
The Salmonella typhimurium strains used in this study were TA1535, TA1537, TA98 and TA100. The Escherichia coli strain used was WP,uvrA.
The strains TA1537 and TA98 are capable of detecting frame shift mutagens, strains TA1535, TA100 and WP,uvrA are capable of detecting base-pair substitution mutage.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
with = S9 microsomefraction of liver homogenate
Test concentrations with justification for top dose:
-Experiment 1 (without S9):
test strains (E. coli, TA1535, TA1535, TA98 and TA100)
concentrations: 15, 50, 150, 500, 1500 and 5000 µg/plate
* TA1537: cytotoxicity was observed and positive control did not produce positive response

-Experiment 1 (without S9): confirmative experiment since the positive control did not produce a positive response in TA1537.
test strains (E. coli, TA1535, TA1535, TA98 and TA100)
concentrations: 15, 50, 150, 500, 1500 and 5000 µg/plate
*TA1537: cytotoxicity was observed

-Experiment 1 (without S9): tested at lower concentration for TA1537
test strains (TA1537)
concentrations: 15, 50, 150, 500, 1500 and 5000 µg/plate (cytotoxicity was observed)
test strains (TA1537)
concentrations: 0.5, 1.5 and 5 µg/plate

-Experiment 2 (with S9)
test strains (E. coli, TA1535, TA1535, TA98 and TA100)
concentrations: 15, 50, 150, 500, 1500 and 5000 µg/plate
*TA1537: no cytotoxicity was observed, but positive control did not produce positive response

-Experiment 2 (with S9): confirmative experiment since the positive control did not produce a positive response in TA1537.
test strains (E. coli, TA1535, TA1535, TA98 and TA100)
concentrations: 15, 50, 150, 500, 1500 and 5000 µg/plate

-Experiment 2 (with S9):
test strains (TA1537)
concentrations: 15, 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulphoxide
- Justification for choice of solvent/vehicle: the test substance is soluble in DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -N-ethyl-N-nitro-N-nitrosoguanidine -Sodium azide -2-nitrofluorene -9-aminoacridine -Other: 2-Aminoanthracene (2AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Preincubation period: not applicable.
- Exposure duration: approximately 65 hours incubation at 37 °C
- Selection time (if incubation with a selection agent): Not applicable.
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable.

NUMBER OF REPLICATIONS: Triplicate.

NUMBER OF CELLS EVALUATED: 2x109.

OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable.
- Determination of endoreplication: not applicable.
Evaluation criteria:
A test substance is considered positive if for any strain, a significant increase over the negative control in the number of revertants per plate was observed which was concentration-dependent. A significant increase was a two-fold increase when the background was 50 revertants/plate or greater; a three-fold increase when the background was between 10 and 49 revertants/plate; and a four-fold increase when the background was less than 10 revertants/plate.
Statistics:
No formal hypothesis testing was done.

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:
other: YES to TA1537 without S9. NO to TA98, TA100, TA1535 and E. Coli without and with S9, TA1537 with S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
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

At the concentrations tested and under the conditions of this assay, the test material was considered non-mutagenic.
Executive summary:

Methods.OS81067 was tested in the Salmonella typhimuriurn reverse mutation assay with four histidine- requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).

Results.Test substance was soluble in dimethyl sulfoxide, and it was tested up to concentrations of 5000 ug/plate in the absence (repeated three times) and presence (repeated three times) of S9-mix in the strains TA1535, TA1537, TA98, TA100 and E.Coli WP2.

Experiment 1 (without S9):

(1) No biologically relevant increase in the number of revertants was observed at all dose levels tested up to the dose level of 5000 ug/plate in all strains in the absence of S9. However, severe cytotoxicity was observed in TA1537. Also, the positive control group in this strain failed to produce positive response.

(2) The test was repeated at the same dose range (15, 50, 150, 500, 1500 and 5000 ug/plate), no mutagenic effects were found in all tested strains; cytotoxicity was observed in TA1537 strain

(3) TA1537 alone was tested at lower concentration range (0.5, 1.5 and 5 ug/plate) to avoid cytotoxicity. No mutagenic effect was observed in this test.

Experiment 2 (with S9):

(1) No biologically relevant increase in the number of revertants was observed at all dose levels tested up to the dose level of 5000 ug/plate in all strains in the presence of S9, no cytotoxicity was observed in any test strains, but positive control group in this strain failed to produce positive response in TA1537.

(2) The test was repeated at the same dose range (15, 50, 150, 500, 1500 and 5000 ug/plate) in the presence of S9, no mutagenic effects were found in all tested strains;

(3) TA1537 alone was tested at concentrations of 15, 50, 150, 500, 1500 and 5000 ug/plate. No mutagenic effect was observed in this test.

Based on the results of this study it is concluded that the test substance is non-mutagenic in tester strains TA1535, TA1537, TA98, TA100 and E.Coli WP2, with/without metabolic activation.