sec-butyl chloroformate

Genetic toxicity: in vitro

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: Guideline conform GLP study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

and OECD Guideline 472

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

sec.-Butylchloroformate

IUPAC Name:

sec.-Butylchloroformate

Constituent 2

Reference substance name:

sec-butyl chloroformate

EC Number:

241-475-6

EC Name:

sec-butyl chloroformate

Cas Number:

17462-58-7

Molecular formula:

C5H9ClO2

IUPAC Name:

butan-2-yl carbonochloridate

Constituent 3

Reference substance name:

96/546

IUPAC Name:

96/546

Details on test material:

- Name of test material (as cited in study report): sec.-Butylchlorformiat (sec.- Butylchlorformate)- Physical state: colorless liquid - Analytical purity: 99.5 % (by GC)- Lot/batch No.: Vers.121/V - Expiration date of the lot/batch: not stated - Stability under test conditions: verified by reanalysis- Storage condition of test material: -20°C

Method

Target gene:

Salmonella typhimurium: (his-) E. coli: (Trp-)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

20 µg - 5,000 µg/plate (SPT)125 µg - 2,000 µg/plate (PIT)

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: see free text

Details on test system and experimental conditions:

Experiment 1: Standard plate test (SPT)Test tubes containing 2 ml soft agar kept in a water bath at 45°C, and remaining components added in the following order: 0 .1 ml test solution or vehicle 0 .1 ml bacterial suspension 0 .5 ml S-9 mix (in tests with metabolic activation) or 0.5 ml phosphate buffer (in tests without metabolic activation). After mixing, the samples are poured onto Vogel-Bonner agar plates. Experiment 2: Preincubation assay (PIT)0.1 ml test solution or vehicle, 0.1 ml bacterial suspension and 0.5 ml S-9 mix are incubated at 37°C for the duration of 20 minutes. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the Vogel-Bonner agar plates. Experiment 1&2 In each experiment 3 test plates per dose or per control used; after incubation at 37°C for 48 hours in the dark, the bacterial colonies ( his+ revertants) are counted. Positive control: with metabolic activation: 2.5 μg/plate 2-aminoanthracene for each strain; without metabolic activation: 5 μg/plate N-methyl-N'-nitro-N-nitrosoguanidine for TA 100 and TA 1535, 10 μg/plate 4-nitro-o-phenylendiamine for TA 98, 100 μg/plate 9-aminoacridine chloride monohydrate for TA 1537 and 10 µg/plate N-Ethyl-N-nitro-N-nitrosoguanidine for E. coli WP2 uvrA, all substances were dissolved in DMSO. The titer was determined and in regularly measurements the strain characteristics were checked. Sterility control was performed.

Evaluation criteria:

Positive results - doubling of the spontaneous mutation rate (control) - dose-response relationship - reproducibility of the result

Statistics:

Mean and standard deviation calculated in result tables. No further data.

Results and discussion

Test results

Test results 1

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:

cytotoxicity

Remarks:

A bacteriotoxic effect was observed depending on the strain and test conditions at doses > 2,500 µg/plate (SPT) or 1,000 µg/plate (PIT).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A bacteriotoxic effect was observed depending on test conditions at doses > 2,500 µg/plate (SPT) or 1,000 µg/plate (PIT).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Negative and positive controls gave expected results.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Mutagenicity

Standard plate test (20 - 5000 µg/plate)
Strain	Metabolic activation system	Replicates	maximum revertant factor	dose dependency	Assessment
TA 98	no	3	0.9	no	negative
	yes	3	0.8	no	negative
TA 100	no	3	1.0	no	negative
	yes	3	1.0	no	negative
TA 1535	no	3	1.1	no	negative
	yes	3	1.0	no	negative
TA 1537	no	3	0.8	no	negative
	yes	3	0.8	no	negative
WP2 uvr A	no	3	0.8	no	negative
	yes	3	0.9	no	negative
Preincubation test (125 - 2000 µg/plate)
Strain	Metabolic activation system	Replicates	maximum revertant factor	dose dependency	Assessment
TA 98	no	3	1.0	no	negative
	yes	3	0.9	no	negative
TA 100	no	3	1.0	no	negative
	yes	3	1.0	no	negative
TA 1535	no	3	0.9	no	negative
	yes	3	1.0	no	negative
TA 1537	no	3	0.9	no	negative
	yes	3	1.1	no	negative
WP2 uvr A	no	3	1.0	no	negative
	yes	3	0.8	no	negative

Applicant's summary and conclusion

Executive summary:

The study is conducted according to the OECD Guideline 471, 472, respectively and is reliable with out any restriction.

sec.-Butylchloorformate was tested in the standard plate test as well as in the preincubation assay with and without metabolic activation (MA) in S. typhimurium TA98, TA100, TA1535 and TA1537 as well as in E. coli WP2uvrA at dose levels of 20 - 5000 µg/plate and 125 - 2000 µg/plate, respectively. No increase in the number of revertants was detected in any strain with and without MA. Vehicle controls and positive controls were valid. Cytotoxicity was found depending on the strain and MA in concentrations above 1000 µg/plate. No precipitation was seen at any dose level.

Conclusion:   

According to the results of the present study, the test substance sec.-Butylchloroformate is not mutagenic in the Salmonella typhimurium/ Escherichia coli reverse mutation assay under the experimental conditions chosen here.