2-ethylhexyl 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:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexyl chloroformate

EC Number:

246-278-9

EC Name:

2-ethylhexyl chloroformate

Cas Number:

24468-13-1

Molecular formula:

C9H17ClO2

IUPAC Name:

2-ethylhexyl carbonochloridate

Details on test material:

- Name of test material (as cited in study report): 2-Ethyihexylchiorformiat
- internal Test substance No .: 99/34-1
- Lot/batch No.: Vers. Nr. 175/Y
- Date of manufacture : January 14, 1999
- Analytical purity: 99,5% (GC)
- Appearance, consistency: Colorless, liquid
- Storage: Room temperature

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:

Standard S9-Mix

Test concentrations with justification for top dose:

Experiment 1: 0; 20; 100 ; 500 ; 2,500 and 5,000 µg/plate
experiment 2&3: 0; 0 .8 ; 4; 20; 100 and 500 pg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: soulability

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

all strains with S9 mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine ( MNNG)

Remarks:

TA1535; TA 100 without S9 mix

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine (NOPD)

Remarks:

TA 98 without S9 mix

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 without S9 mix

Controls 5

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

E . coli WP2 uvrA without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION
Standard plate test and preincubation test

DURATION
- Preincubation period: 20 min
- Expression time : 48 - 72 hours

SELECTION AGENT (mutation assays):
The Salmonella strains are checked for the following characteristics at regular intervals: deep rough character (rfa) ; UV sensitivity (o uvrB); ampicillin resistance (R factor plasmid). E . coli WP2 uvrA is checked for UV sensitivity. Histidine and tryptophan auxotrophy is automatically checked in each experiment via the spontaneous rate.

NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
Backgound

Evaluation criteria:

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

Statistics:

mean and SD of replicates

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

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not reported
- Effects of osmolality: not reported
- Evaporation from medium: not expected (vapour pressure 0.4 mbar)
- Water solubility: not reported
- Precipitation: No
- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
Experiment1 was the screening study

COMPARISON WITH HISTORICAL CONTROL DATA:
The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A bacteriotoxic effect (reduced his or trp" background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed depending on the strain and test conditions from about 100 pg - 500 pg/plate onward .

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Toxicity was not observed in cells treated with > = 2500 micrograms/plate (standard plate test) and >= 100 micrograms/plate (preincubation test). The test material did not precipitate. Positive controls induced at least a 6-fold increase in mutants.

Salmonella:

Standard plate test: There was no mutagenic effect of test material in the presence or absence of S-9. In the absence of S-9, the average numbers of mutants in controls experiments in strains TA98, TA100, TA1535 and TA1537 in both experiments were 23 and 27, 127 and 142, 19 and 21, and 10 and 12, respectively. The average numbers of mutants in treated strains TA98, TA100, TA1535 and TA1537 in both experiments ranged from 2-24,2-145, 5-22 and 1-9, respectively. In the presence of S-9, the average numbers of mutants in controls in strains TA98, TA100, TA1535 and TA1537 in both experiments were 34 and 36, 133 and 144, 19 and 20 and 10 and 11, respectively. The average numbers of mutants in treated strains TA98, TA100, TA1535 and TA1537 in both experiments with S-9 ranged from 1-30, 3-156, 1-20 and 1-13, respectively. 

Preincubation test: There was no mutagenic effect of test material in the presence or absence of S-9. In the absence of S-9, the average numbers of mutants in controls in strains TA98, TA100, TA1535 and TA1537 were 26, 122, 19 and 11, respectively. The average numbers of mutants in treated strains TA98, TA100, TA1535 and TA1537 ranged from 19-30, 118-127, 10-15 and 6-10, respectively. In the presence of S-9, the average numbers of mutants in controls in strains TA98, TA100, TA1535 and TA1537 were 32, 119, 19 and 10, respectively. The average numbers of mutants in treated strains TA98, TA100, TA1535 and TA1537 with S-9 ranged from 26-33, 104-132, 14-17 and 6-14, respectively.

E.Coli.

Standard plate test: There was no mutagenic effect of test material in the presence or absence of S-9. In the absence of S-9, the average numbers of mutants in controls in the 2 experiments were 23 and 29. The averagenumber of mutants in treated cells in the 2 experiments ranged from 14-27. In the presence of S-9, the average numbers of mutants in controls in the 2 experiments were 34 and 35. The average number of mutants in treated cells (both experiments) ranged from 14-36. 

Preincubation test: There was no mutagenic effect of test material in the presence or absence of S-9. In the absence of S-9, the average number of mutants in the control was 28. The average number of mutants in treated cells ranged from 32-35. In the presence of S-9, the average number of mutants in the control was 25. The average number of mutants intreated cells ranged from 18-28.

Applicant's summary and conclusion

Conclusions:

The test substance 2-Ethylhexylchlorformiat is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen here.

Executive summary:

2-Ethyihexylchiorformiat was tested for its mutagenic in an Ames Test. The test strains waer TA1535, TA 100, TA 1537, Ta 98 and E.coli WP2 uvrA in a dose range of 0.8 µg - 5000 µg/plate (SPT) and 0.8 µg - 500 µg/plate (PIT). Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (Aroclor-induced rat liver S-9 mix). No precipitation of the test substance was found. A bacteriotoxic effect was observed depending on the strain and test conditions from about 100 µg - 500 µg/plate onward. An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system. According to the results of the present study, the test substance 2-Ethylhexylchlorformiat is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen here .