Propiononitrile

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22nd September 1999 - 12th October 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

There were no significant deviations from the aforementioned regulations or the signed protocol that would affect the integrity of the study or the interpretation of the test results.

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

yes

Remarks:

There were no significant deviations from the aforementioned regulations or the signed protocol that would affect the integrity of the study or the interpretation of the test results.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Butyronitrile

EC Number:

203-700-6

EC Name:

Butyronitrile

Cas Number:

109-74-0

Molecular formula:

C4H7N

IUPAC Name:

Butyronitrile

Test material form:

liquid

Details on test material:

CAS RN: 109-74-0
EC No: 203-700-6

Specific details on test material used for the study:

Lot D-7
Acc. No. 907253
Physical Description: transparent colorless liquid
Date Received: 09/21/99

Method

Target gene:

his, uvrB genes.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: In addition to the histidine operon, the strain had two additional mutations to enhance the sensitivity to mutagenic compounds. The rfa mutation and a deletion of the urv gene. TA98 and TA100 contain R-factor plasmid (pKM101).

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Liver microsomal enzyme reaction mix (S9 Mix) from Aroclor induced male Sprague-Dawley rats.

Test concentrations with justification for top dose:

Range finding study concentrations (ug/plate): 6.67, 10, 33.3, 66.7, 100, 333, 667, 1000, 3330, 5000.
Mutagenicity assay concentrations (ug/plate): 100, 330, 1000, 3330, 5000.
The actual mutagenicity study concentrations were derived from the range finding mutagenicity study. Range finding study conducted using tester strains TA100 and WP2urvA(pKM101) in presence and absence of S9 mix, where no cytotoxicity was observed with either of the strains.

Vehicle / solvent:

Dimethyl-sulphoxide

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 (with S9 mix); ICR-191 (without S9 mix).

Rationale for test conditions:

Study conducted as per OECD guidelines.

Evaluation criteria:

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. pKM 101 Plasmid
To demonstrate the presence of the R-factor plasmid, pKM101, cultures of tester strains TA98 and TA100 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
TA100 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, TA100, 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 TA1535 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 results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

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

Test results 2

Key result

Species / strain:

S. typhimurium 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

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

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

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

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

Test results 5

Key result

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

Additional information on results:

Dose range-finding study.

Doses tested in the mutagenicity assay were selected based on the results of the dose range-finding study conducted on the substance using tester strains TA100 and WP2uvrA(pKM101) in both the presence and absence of S9 mix (one plate per dose). Ten doses of the substance, ranging from 5,000 to 6.67 μg per plate. No cytotoxicity was observed with either tester strain in either the presence or absence of S9 mix as evidenced by a normal background lawn and no decrease in the number of revertants per plate. No substance precipitate was observed on the plates at any of the doses tested.

Mutagenicity assay.

The results of the dose range-finding study were used to select the five doses tested in the mutagenicity assay. The doses tested were 5,000, 3,330, 1,000, 333, and 100 μg per plate in both the presence and absence of S9 mix. All data were acceptable and no positive increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix.

Remarks on result:

other: Substance did not cause a positive increase in the mean number of revertants per plate.

Any other information on results incl. tables

Mutagenicity assay results: Summary 1.

With microsomal activation

Concentration (ug/plate)	TA 98  Mean Rvertants/plate	TA 100 Mean Rvertants/plate	TA 1535 Mean Rvertants/plate	TA 1537   Mean Rvertants/plate	WP2urvA (pKM101) Rvertants/plate
100	14	109	12	7	120
333	16	111	8	9	126
1000	18	104	11	9	138
3330	18	108	12	11	166
5000	17	86	10	7	149
Solvent control	16	101	15	6	118
Positive control	915	731	143	223	1487

Without microsomal activation:

Concentration (ug/plate)	TA 98 Mean Rvertants/plate	TA 100 Mean Rvertants/plate	TA 1535 Mean Rvertants/plate	TA 1537   Mean Rvertants/plate	WP2urvA (pKM101) Mean Rvertants/plate
100	9	83	12	5	111
333	6	80	8	7	102
1000	9	88	12	4	99
3330	13	100	10	7	107
5000	9	87	7	6	95
Solvent control	10	81	8	6	120
Positive control	220	463	500	634	1946

Mutagenicity assay results: Summary 2.

With microsomal activation:

Concentration (ug/plate)	TA 98  Mean Rvertants/plate	TA 100 Mean Rvertants/plate	TA 1535 Mean Rvertants/plate	TA 1537 Mean Rvertants/plate	WP2urvA (pKM101) Mean Rvertants/plate
100	22	107	7	8	214
333	22	94	10	10	206
1000	27	108	25	8	207
3330	18	96	20	10	211
5000	22	99	25	11	206
Solvent control	22	98	16	6	201
Positive control	853	852	155	177	887

Without microsomal activation:

Concentration (ug/plate)	TA 98 Mean Rvertants/plate  (with/without S9 mix)	TA 100 Mean Rvertants/plate  (with/without S9 mix)	TA 1535 Mean Rvertants/plate  (with/without S9 mix)	TA 1537   Mean Rvertants/plate  (with/without S9 mix)	WP2urvA (pKM101) Mean Rvertants/plate (with/without S9 mix)
100	15	73	9	6	125
333	11	74	11	4	132
1000	11	81	10	4	151
3330	6	82	10	6	163
5000	7	93	8	4	209
Solvent control	15	93	7	4	153
Positive control	199	608	525	477	1668

Backgrownd lawn was evaluated for all controls and the substance, which reported as in the normal range.

Applicant's summary and conclusion

Conclusions:

The substance showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Executive summary:

The potential of the substance to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium: TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA was evaluated in the presence or absence of an exogenous mammalian metabolic activation system (S9). The study was conducted according to EEC Annex V Guideline number B.14, "Other Effects-Mutagen Salmonella typhimurium-Reverse Mutation Assay", and Guideline number B.13, Other Effects-Mutagenicity, Escherichia co-Reverse Mutation Assay under GLP assurances. The concentrations of the substance employed were 100, 333, 1000, 3330, and 5000 µg/plate (in triplicate). The concentrations were selected on a basis of a dose-range finding study. None of the concentrations tested caused toxicity. No precipitate was observed at the maximum concentration tested. All criteria for a valid test were met. No positive responses were induced in any of the strains. Based on the results of this study, the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.