Reaction mass of 3-O-acetyl-1,5-anhydro-4-butyl-2,4-dideoxy-2-methyl-D-xylitol and (2R,3S)-2-methyloctane-1,3-diyl diacetate

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:

28 August, 2002 - 23 September, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This information is used for read across to Jessemal.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2000)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Type:

Constituent

Test material form:

liquid

Method

Target gene:

- S. typhimurium: Histidine gene

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced with Aroclor 1254

Test concentrations with justification for top dose:

- Dose range finding test:
Direct plate assay and pre-incubation assay: TA 100 (without and with S9): 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate

- Experiment 1 (direct plate):
Due to cytotoxicity, the following dose levels were used:
TA 1535, TA 1537, TA 98 (without and with S9): 10, 33, 100, 333, 1000 and 3330 µg/plate
TA 102 (with S9): 10, 33, 100, 333, 1000 and 3330 µg/plate
TA 102 (without S9): 33, 100, 333, 1000, 3330 and 5000 µg/plate

- Experiment 2 (preincubation):
Due to cytotoxicity, the following dose levels were used:
TA 1535, TA 1537, TA 98 and TA 102 (without and with S9): 3, 10, 33, 100, 333 and 1000 µg/plate

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: the test substance was found to be soluble in DMSO up to 5000 µg/plate.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

(100 µL/plate)

Positive controls:

yes

Positive control substance:

other: see section "Any other information on materials and methods incl. tables"

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Experiment 1: in agar (plate incorporation)
- Experiment 2 (independent repeat): preincubation

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain (in all experiments)

DETERMINATION OF CYTOTOXICITY
- Method: on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one repeated experiment.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at the concentrations of 3330 and 5000 µg/plate in experiment 1 only.

RANGE-FINDING/SCREENING STUDIES:
- In strain TA100 toxicity was observed at 3330 µg/plate and above, both in the absence and presence of S9 in the direct plate assay. In the pre-incubation assay, in strain TA100 toxicity was observed at 333 or 1000 µg/plate and above in the absence or presence of S9 respectively.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment 1 in all strains toxicity was observed, except in tester strain TA1535 (with S9) and TA1537 (without S9). In experiment 2, toxicity was observed in all tester strains.

Applicant's summary and conclusion

Conclusions:

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay performed according to OECD 471 (1997) guideline and GLP principles.

Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) guideline and according to GLP principles. The test was performed in two independent experiment. At first a direct plate assay was performed and secondly a pre-incubation assay, both in the absence and presence of S9-mix. The dose levels were selected based on observed cytotoxicity in dose range finding tests (>= 333 ug/plate). Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the five S. typhimurium tester strains (TA1535, TA1537, TA98, TA100 and TA102), both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.