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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted in a GLP facility under OECD guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Butyronitrile
EC Number:
203-700-6
EC Name:
Butyronitrile
Cas Number:
109-74-0
Molecular formula:
C4H7N
IUPAC Name:
butyronitrile
Details on test material:
Test material was supplied as a liquid

Method

Target gene:
His gene and uvr B genes
Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Liver microsomes from Arochlor 1254 treated rats
Test concentrations with justification for top dose:
5000, 3330, 1000, 333 and 100 ug/plate
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
other: 2-aminoanthracene, ICR-191
Evaluation criteria:
Criteria for a Valid Assay
The following criteria were used to determine a valid assay:
1. Tester Strain Integrity: Salmonella typhimurium
a. rfa Wall Mutation
To demonstrate the presence of the rfa wall mutation, tester strain
cultures exhibited sensitivity to crystal violet.
b. pKMlOl Plasmid
To demonstrate the presence of the R-factor plasmid, PKM101,
cultures of tester strains TA98 and TAl00 exhibited resistance to ampicillin.
c. Characteristic Number of Spontaneous Revertants
To demonstrate the requirement for histidine, the tester strain
cultures exhibited a characteristic number of spontaneous revertants per plate when plated along
with the vehicle under selective conditions. The acceptable ranges for the vehicle controls were
as follows:
TA98 8 - 60
TAl00 60 - 240
TA1535 4 - 45
TA1537 2 - 25

Criteria for a Positive Response
Once the criteria for a valid assay had been met, responses observed in the assay
were evaluated as follows:
Tester Strains TA98, TAI00, and WP2uvrA(PKM 101)
For a test article to be considered positive, it had to produce at least a
2-fold increase in the mean revertants per plate of at least one of these tester strains over the
mean revertants per plate of the appropriate vehicle control. This increase in the mean number of
revertants per plate had to be accompanied by a dose response to increasing concentrations of the
test article.
2. Tester Strains TAI535 and TA1537
For a test article to be considered positive, it had to produce at least a
3-fold increase in the mean revertants per plate of at least one of these tester strains over the
mean revertants per plate of the appropriate vehicle control. This increase in the mean number of
revertants per plate had to be accompanied by a dose response to increasing concentrations of the
test article.

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
valid
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

The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that under the conditions of this study, the test article did not cause a positive increase in the mean number of revertants per plate with any of the tester strains in either the presence or absence of microsomal enzymes prepared from Aroclor™-induced rat liver (S9).
Executive summary:

The test article was investigated for mutagenic activity in the Salmonella-Escherichia coli/ Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay. This assay evaluated the test article and/or its metabolites for their ability to induce reverse mutations at the histidine locus in the genome of specific Salmonella typhimurium tester strains and at the tryptophan locus in an Escherichia coli tester strain both in the presence and absence of an exogenous metabolic activation system of mammalian microsomal enzymes derived from Aroclor™ 1254 -induced rat liver (S9). The doses tested in the mutagenicity assay were selected based on the results of a dose

rangefinding study using tester strains TA 100 and WP2uvr A(pKM 101) and ten doses of test article ranging from 5,000 to 6.67 ug per plate, one plate per dose, both in the presence and absence of S9 mix. The tester strains used in the mutagenicity assay were Salmonella typhimurium TA 98, TA l00, TA l535, and TA 1537 tester strains and Escherichia coli tester strain WP2uvrA(pKM101). The assay was conducted with five doses of test article in both the presence and absence of S9 mix along with concurrent vehicle and positive controls using three plates per dose. The doses tested were 5,000, 3,330, 1,000, 333, and 100 ug per plate in both the presence and absence of S9 mix. The results of the initial mutagenicity assay were confirmed in an independent experiment.

The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that under the conditions of this study, n-butyronitrile did not cause a positive increase in the mean number of revertants per plate with any of the tester strains in either the presence or absence of microsomal enzymes prepared from Aroclor™ 1254 -induced rat liver (S9).

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