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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:
Apr 02 -Apr 04, 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was performed before Good Laboratory Practice (GLP) regulations (revised in 1997, ENV/MC/CHEM(98)17) and is thus not a GLP study. The method followed that described in the OECD Guidelines for Testing of Chemicals (Adopted: 4 April 1984) No 471 "Bacterial Reverse Mutation Test".

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
- single series only
- purity of test substance is missing
- ammount of 10% rat liver homogenate (S9 mix) with standard co-factors used is missing
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Chemical Name: CCP-V2-1

Method

Target gene:
HIS operon (S. thyphimurium)
TRY operon (E. coli)
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535
Details on mammalian cell lines (if applicable):
his G 46, uvrB, rfa
Additional strain characteristics:
other: mutations in the histidine operon
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate (S9 mix) with standard co-factors with metabolic activation (Aroclor)
Species / strain:
S. typhimurium TA 1537
Details on mammalian cell lines (if applicable):
his C 3076, uvrB, rfa
Additional strain characteristics:
other: mutations in the histidine operon
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors
Species / strain:
S. typhimurium TA 98
Details on mammalian cell lines (if applicable):
his D 3052, uvrB, rfa + R-factor
Additional strain characteristics:
other: mutations in the histidine operon
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors
Species / strain:
S. typhimurium TA 100
Details on mammalian cell lines (if applicable):
his G 46, uvrB, rfa + R-factor
Additional strain characteristics:
other: mutations in the histidine operon
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors
Species / strain:
E. coli WP2
Details on mammalian cell lines (if applicable):
his C 3076, uvrB, rfa
Additional strain characteristics:
other: mutations in the tryptophan operon
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors
Test concentrations with justification for top dose:
1. Series: 0, 150, 500, and 1500 µg/plate
Vehicle:
The test substance was dissolved up to 50 mg/ml in acetone, and was used for the assay immediately after diluting with the same solvent.
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Positive controls:
yes
Positive control substance:
furylfuramide
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9
Positive controls:
yes
Positive control substance:
9-aminoacridine
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9
Details on test system and conditions:
Test procedures :
The test was performed by the plate incorporation method.
In a test tube, 2 ml of molten top agar, 0.1 ml of the growth culture of a test strain, 0.1 ml of the test substance solu¬tion and 0.5 ml of the phosphate buffer for the direct method or the S9 mix for the metabolic activation method, were mixed and poured over the surface of a minimal agar plate.

For the negative and the positive control tests, each 0.1 ml of solvent or positive substance solution was added respectively instead of adding the test substance solution.

All plates were incubated for 48 hours at 37°C. At the end of the incubation period, revertants appeared on each plate were counted, and mean of two plates counts was calculated.
Evaluation criteria:
The results were judged to be positive when the revertants were twice or more that of the negative control, and when dose dependency or reproducibility were observed in the in-crease of revertants at least in one strain.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
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 with and without metabolic activation

With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under
the experimental conditions described.
Executive summary:

Purpose The purpose of this in vitro reverse gene mutation test is to identify agents that cause mutations in bacteria cells thus providing information on possible health hazards for the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in man.

Study Design

Salmonella typhimurium and Escherichia coli strains were tested in accordance with the plate incorporation method described by Ames et al. (1975) with and without metabolic activation at doses between 150 and 1500 µg/plate.

Results

There were no increasing number of revertants observed for any strains and any concentration of the test item. The positive control substances increased the number of mutants twice or more the negative control in each strains. These counts were within the range of historical control data, thus the sensitivity of the strains and the stability of the positive control substance were shown. The revertants of five strains, Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA, did not increase at any doses.

Conclusions

It is concluded that the test substance has no mutagenic activity in this test system.