2-isopropyl-N-hydroxyethyl 1,3-oxazolidine;methyl carbonato-N-ethyl-2-isopropyl-1,3-oxazolidine;reaction mass of: carbonato-bis-N-ethyl-2-isopropyl-1,3-oxazolidine

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:

from 1998-03-09 to 1998-05-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted: 1983

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

cited as: Directive 92/69/EEC method B14 of 1993

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

429-990-6

EC Name:

-

Molecular formula:

Not applicable as multi-const. substance. Please refer to IUCLID section 1.2 for details on constituents.

IUPAC Name:

2-[(2R)-2-(propan-2-yl)-1,3-oxazolidin-3-yl]ethan-1-ol; 2-[(2R)-2-(propan-2-yl)-1,3-oxazolidin-3-yl]ethyl 2-[(2S)-2-(propan-2-yl)-1,3-oxazolidin-3-yl]ethyl carbonate; 2-[(2S)-2-(propan-2-yl)-1,3-oxazolidin-3-yl]ethan-1-ol; bis{2-[(2S)-2-(propan-2-yl)-1,3-oxazolidin-3-yl]ethyl} carbonate; methyl 2-[(2R)-2-(propan-2-yl)-1,3-oxazolidin-3-yl]ethyl carbonate; methyl 2-[(2S)-2-(propan-2-yl)-1,3-oxazolidin-3-yl]ethyl carbonate

Test material form:

liquid: viscous

Method

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:

The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was prepared from male Sprague Dawley rats induced with Aroclor 1254.

Test concentrations with justification for top dose:

Concentration range in the main test (with and without metabolic activation): 8, 40, 200, 1000, 5000 µg/plate
Concentration range in the main test (with and without metabolic activation): 8, 40, 200, 1000, 5000 µg/plate

Vehicle / solvent:

- Solvent: Dimethylsulfoxide

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: without S9: TA102 with Glutaraldyhyde; with S9: TA102 with 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and pre-incubation

DURATION for plate incorporation (Experiment 1)
- Exposure duration: 72h

DURATION for pre-incubation method (Experiment 2)
- Pre-incubation period: incubated for 2 hour at 37 °C,
- Exposure duration: 72h

NUMBER OF REPLICATIONS: 3
Negative (solvent) controls were included in each assay, in quintuplicate without and with S-9.

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies per plate and/or by a thinning or disappearance of the bacterial background lawn

Evaluation criteria:

The test item was considered to be mutagenic if :
1. the assay was valid
2. Dunnetts test gave a significant response, and the data set showed a significant dose-correlation
3. the positive responses described in 2) were reproducible.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

>5000µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Precipitation: no

COMPARISON WITH HISTORICAL CONTROL DATA:
The observed reversion rates were not different from the historical control range.
Other: Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

Incozol LV did not induce mutation in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102) when tested at concentrations up to 5000 µg/plate, in the absence and presence of a rat liver metabolic activation system (S-9).

Executive summary:

A study was conducted according to OECD TG 471 and Directive 92/69/EEC method B.14 to assess the mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments. An initial toxicity range-finder experiment was carried out in strain TA100 only, using final concentrations of Incozol LV at 8-5000 µg/plate, plus negative (solvent) and positive controls. Following these range-finder treatments, no evidence of toxicity was observed. In order to assess the reproducibility of an increase in revertant numbers observed in the range-finder experiment, repeat treatments of strain TA100 in the absence of S-9 were performed alongside the remaining strains in Experiment 1. All Experiment 1 treatments were performed using the same dose range as employed in the range-finder experiment. Following these Experiment 1 treatments, once again no evidence of toxicity was observed. Experiment 2 retained 5000 µg/plate as the maximum test dose and employed a narrowed dose range, in order to more closely investigate those doses considered most likely to induce any mutation. Treatments in the presence of S-9 included a pre-incubation step, in order to increase the range of mutagenic chemicals that could be detected using this assay system. Additional strain TA102 treatments in the presence of S-9 were included using the plate-incorporation methodology, in order to more thoroughly investigate a small increase in revertant numbers seen in Experiment 1. Evidence of toxicity was observed following preincubation treatments of all strains performed at the higher test doses, in the presence of S-9. No toxic signs were observed following any treatments of the test strains performed in the absence of S-9, or following treatments of strain TA102 in the presence of S-9 using plate incorporation methodology. Negative (solvent) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments. No Incozol LV treatment of any of the test strains produced an increase in revertant numbers sufficient to be considered as clearly indicative of mutagenic activity. It was concluded that Incozol LV did not induce mutation in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102) when tested under the conditions employed for this study, which included treatments at concentrations up to 5000 µg/plate, in the absence and presence of a rat liver metabolic activation system (S-9).