4-tert-butylcyclohexyl 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

p.t. Butylcyclohexylchlorformate

IUPAC Name:

p.t. Butylcyclohexylchlorformate

Constituent 2

Reference substance name:

4-tert-butylcyclohexyl chloroformate

EC Number:

255-670-9

EC Name:

4-tert-butylcyclohexyl chloroformate

Cas Number:

42125-46-2

Molecular formula:

C11H19ClO2

IUPAC Name:

4-tert-butylcyclohexyl carbonochloridate

Constituent 3

Reference substance name:

96/615

IUPAC Name:

96/615

Details on test material:

- Name of test material (as cited in study report): p.t. Butylcyclohexylchlorformiat (p.t. Butylcyclohexylchlorformate)
- Physical state: colorless liquid
- Analytical purity: 97,3 %
- Lot/batch No.: Vers.167/V
- Expiration date of the lot/batch: not stated
- Stability under test conditions: not stated
- 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

Test concentrations with justification for top dose:

20-5000 µg/plate (standard plate test)
4-1000 µg/plate (pre-incubation test)

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
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
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. S ubsequently, 2 ml of soft agar is added
and, after mixing, the samples are poured onto the Vogel-Bonner agar plates.

Experiment1&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 9-aminoacridine chloride monohydrate for TA 1537 and 10 µg/plate N-tylN-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 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 from about 500 - 1,000 µg/plate onward.

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:

from about 500 µg/plate onward.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Negative and positive controls gave expected results. Mutation rate was with in the range of the historical control data

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Standard plate Test (range finder/pre-experiment)

Strain	Metabolic activation	Concentration [µg/plate]	Replicates	Max. factor of revertants	Dose dependency
TA 98	No yes	20-5000 20-5000	3 3	1.0 1.0	No declining (Cytotox.)
TA 100	No yes	20-5000 20-5000	3 3	1.0 1.0	No No
TA 1535	No yes	20-5000 20-5000	3 3	1.0 0.9	No declining (Cytotox.)
TA 1537	No yes	20-5000 20-5000	3 3	1.0 0.9	No No
E.Coli WP2 uvrA	No yes	20-5000 20-5000	3 3	1.1 0.9	No No

Standard plate test

Strain	Metabolic activation	Concentration [µg/plate]	Replicates	Max. factor of revertants	Dose dependency
TA 98	No yes	62.5-1000 62.5-1000	3 3	0.8 0.9	declining (Cytotox.) declining (Cytotox.)
TA 100	No yes	62.5-1000 62.5-1000	3 3	1.0 1.0	No No
TA 1535	No yes	62.5-1000 62.5-1000	3 3	1.0 0.9	No declining (Cytotox.)
TA 1537	No yes	62.5-1000 62.5-1000	3 3	1.1 0.8	No declining (Cytotox.)
E.Coli WP2 uvrA	No yes	62.5-1000 62.5-1000	3 3	1.1 0.9	No declining (Cytotox.)

Pre-incubation test

Strain	Metabolic activation	Concentration [µg/plate]	Replicates	Max. factor of revertants	Dose dependency
TA 98	No yes	4 -1000 4 -1000	3 3	0.9 0.9	declining (Cytotox.) declining (Cytotox.)
TA 100	No yes	4-1000 4-1000	3 3	0.9 1.0	declining (Cytotox.) declining (Cytotox.)
TA 1535	No yes	4-1000 4-1000	3 3	0.9 0.8	declining (Cytotox.) declining (Cytotox.)
TA 1537	No yes	4-1000 4-1000	3 3	0.8 0.8	declining (Cytotox.) declining (Cytotox.)
E.Coli WP2 uvrA	No yes	4-1000 4-1000	3 3	1.0 0.8	declining (Cytotox.) declining (Cytotox.)

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.

p.t.-Butylcyclohexylchlorformate 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. 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 in concentrations above 500 µg/plate. No precipitation was seen even at any dose level.

Conclusion:  

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