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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
n-Butyronitrile was tested in the standard battery of 3 in vitro assays (Ames, chromosomal aberration and mammalian mutagenicity) with negative results.
Link to relevant study records
Reference
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
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
Target gene:
His gene and uvr B genes
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
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.
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'.
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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Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

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).

The objective of this in vitro assay was to evaluate the ability of EC98-0254, NBN to induce chromosomal aberrations in Chinese hamster ovary (CHO) cells with and without metabolic activation. The test substance was dissolved in dimethylsulfoxide (DMSO) at a concentration of 70.0 mg/mL for the assay. The high dose tested was achieved using a dosing volume of 1.0% (10.0

uL/mL) and the vehicle control cultures were treated with 10.0 uL/mL of DMSO. The high dose in the assay, 700 ug/mL, is 10 mM of the test substance, the recommended high dose for this assay. In the initial chromosomal aberrations assay, the treatment period was for 3.0 hours with and without metabolic activation and cultures were harvested 20.0 hours from the initiation of treatment. Concentrations of 4.77, 6.81, 9.73, 13.9, 19.9, 28.4, 40.5, 57.8, 82.6, 118, 169, 241, 344, 491, and 701 ug/mL were tested with and without metabolic activation. Cultures treated with concentrations of 241, 344, 491, and 701 ug/mL with and without metabolic activation were analyzed for chromosomal aberrations. No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in the cultures analyzed. In a confirmatory chromosomal aberrations assay, the treatment period was for 17.8 hours without metabolic activation and 3.0 hours with metabolic activation, and cultures were harvested 20.0 hours from the initiation of treatment. Concentrations of 27.3, 54.5, 109, 217, 289, 385, 513, and 684 ug/mL were tested without metabolic activation and 217, 289, 385, 513, and 684 ug/mL were tested with metabolic activation. Cultures treated with concentrations of 289, 385, 513, and 684 ug/mL with and without metabolic activation were analyzed for chromosomal aberrations. No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in the cultures analyzed. NBN was considered negative for inducing chromosome aberrations in CHO cells with and without metabolic activation.

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of

Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances. Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the

test item at six dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at six dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence

of metabolic activation. The dose range of test item was selected following the results of a preliminary toxicity test and

was 51.94 to 831 µg/ml in both the absence and presence of metabolic activation for both Experiments 1 and 2.The maximum dose level used in the Mutagenicity Test was the 10 mM limit dose of 831 µg/ml. Precipitate of the test item was not observed at any of the dose levels in the study. The vehicle (solvent) controls had mutant frequency values that were considered acceptable for the

L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either

the first or the second experiment.


Justification for selection of genetic toxicity endpoint
This section does not allow for the selection of multiple tests. n-Butyronitrile was tested in the standard battery of 3 in vitro assays (Ames, chromosomal aberration and mammalian mutagenicity) with negative results.

Justification for classification or non-classification

As negative results were obtained in all three in vitro tests (Ames, chromosomal aberration and mammalian mutagenicity), n-Butyronitrile is not classified for genotoxicity.

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