Oxazolidine, 3-butyl-2-(1-ethylpentyl)-

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:

1995-11-10 to 1995-12-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1993

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:

-

EC Number:

425-660-0

EC Name:

-

Cas Number:

165101-57-5

Molecular formula:

C14H29NO

IUPAC Name:

3-butyl-2-(heptan-3-yl)-1,3-oxazolidine

Method

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- -> his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base-pair substitution (TA 100, TA 1535, TA 102) and frameshift (TA 98, TA 1537).

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: nutrient broth (containing ampicillin for strains TA98 and TA100 and ampicillin and tetracycline for strain TA102)
- Properly maintained: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (Aroclor 1254 induced)

Test concentrations with justification for top dose:

Experiment 1
with and without S9 mix: 8, 40, 200, 1000, 5000 µg/plate

Experiment 2:
without S9 mix: 1000, 2000, 3000, 4000, 5000 µg/plate
with S9 mix (strains TA98, TA1537, TA102): 6.25, 12.5, 25, 50, 100 µg/plate
with S9 mix (TA100): 3.125, 6.25, 12.5, 50 µg/plate
with S9 mix (TA1535): 12.5, 25, 50, 100, 200 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: analytical grade Dimethylformamide (DMF)

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98 without S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100, TA1535 without S9 mix

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 without S9 mix

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Glutaraldehyde (GLU)

Remarks:

TA102 without S9 mix

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (AAN)

Remarks:

TA98, TA100, TA1535 with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION:
For the range-finding study and Experiment 1 a plate incorporation was used. Experiment 2 employed a pre-incubation step in addition to the plate incorporation.

DURATION
- Preincubation period: 1h
- Exposure duration: 3 days
- Expression time (cells in growth medium): 10 h

AMES-TEST
Incozol 2 was tested for mutation in Five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102) using triplicate plates without and with S9. Negative (solvent) controls were included in both assays, in quintuplicate without and with S9. In each experiment, bacterial strains were treated with diagnostic mutagens in triplicate in the absence of S9. The activity of the S9 mix used in each experiment was confirmed by AAN treatments (again in triplicate) of at least one strain in the presence of S9. Platings were achieved as described for the range-finding below.

As the results of the first experiment were negative, treatments in the presence of S9 in Experiment 2 included a pre-incubation step, where the quantities of test article or control solution, were mixed together and incubated for 1 hour at 37°C, before the addition of 2.5 mL molten agar at 46°C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected in the assay.

Following initial Experiment 2 treatments of all the test strains in the presence of S9, a high degree of toxicity was observed, extending over all the test doses. As all test doses were affected by toxicity, these data were not considered acceptable for mutagenicity assessment. These treatments in the presence of S9 were therefore repeated using revised test doses, along with concurrent diagnostic control treatments in the absence of S9. Evidence of toxicity was again observed following these repeat treatments, extending over several test doses in each test strain. Although this toxicity was less severe than that observed following the initial treatments, sufficient treatment doses were again affected to consider the data unsuitable for mutagenicity assessment. Subsequent repeat treatments and diagnostic controls were therefore performed, with maximum test doses further reduced, and the results of these treatments are those presented as the mutagenicity data for Experiment 2 in the presence of S9.

DETERMINATION OF CYTOTOXICITY AND RANGE-FINDING
- Method: Incozol 2 was tested for toxicity in strain TA100, at the concentrations 8, 40, 200, 1000, 5000 µg/plate. Triplicate plates without and with S9 mix were used. Negative (solvent) and positive controls were included in quintuplicate and triplicate respectively without and with S9 mix. These platings were achieved by the following sequence of additions to 2.5 mL molten agar at 46°C:

0.1 mL bacterial culture
0.1 mL test article solution or control
0.5 mL 10% S9 mix or buffer solution

followed by rapid mixing and pouring on to Minimal Davis agar plates. When set, the plates were inverted and incubated at 37°C in the dark for 3 day. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted.

Evaluation criteria:

The test article was considered to be mutagenic if:

1) the assay was valid

2) Dunnett's test gave a significant response (p < 0.01), and the data set showed a significant dose-correlation

3) the positive responses described in 2) were reproducible.

Statistics:

The m-statistic was calculated to check that the data were Poisson-distributed, and Dunnett's test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was checked by linear regression analysis.

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:

cytotoxicity

Remarks:

This was observed in Experiment 2.

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:

cytotoxicity

Remarks:

This was observed in Experiment 2.

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:

cytotoxicity

Remarks:

This was observed in Experiment 2.

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:

cytotoxicity

Remarks:

This was observed in Experiment 2.

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:

cytotoxicity

Remarks:

This was observed in Experiment 2.

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
- Water solubility: The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.
- Precipitation: not observed

RANGE-FINDING/SCREENING STUDIES:
An initial toxicity range-finder experiment was carried out in strain TA100 only, using final concentrations of Incozol 2 at 8-5000 µg/plate, plus negative and positive controls. Following these treatments, no evidence of toxicity was observed, as would normally be indicated by a thinning of the background bacterial lawn, and these results were therefore considered acceptable for use as the TA100 mutagenicity data for Experiment 1. The remaining test strains were treated in Experiment 1 using the same dose range as in the range-finder experiment. As with the range-finder experiment, no evidence of toxicity was observed following any of these test treatments.

COMPARISON WITH HISTORICAL CONTROL DATA: This was done with the solvent controls, which fell within the normal historical ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The increased toxicity that was observed following Experiment 2 treatments in the presence of S9, compared to the other test treatments, was attributed to the conditions occurring during the pre-incubation step (all other treatments employed a plate-incorporation methodology). As volumes of test agent and bacterial cells are pre-incubated prior to the addition of soft agar, and plating out onto minimal agar plates, the effective concentration of test agent that the bacterial cells are exposed to over the pre-incubation period is increased. In this case, this appears to have resulted in toxic effects which were not manifest when using the same treatment doses under plate-incorporation conditions.

Applicant's summary and conclusion

Conclusions:

It was concluded that Incozol 2 did not induce mutation in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), when tested under the conditions employed in this study, which included treatment at concentrations up to either 5000 µg/plate or the lower limit of the toxic range, both in the absence and in the presence of a rat liver metabolic activation system (S9).

Executive summary:

Incozol 2 was assayed for 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 (S9), in two separate experiments. An initial toxicity range-finder experiment was carried out in strain TA100 only, using final concentrations of Incozol 2 at 8-5000 µg/plate, plus solvent and positive controls. Following these treatments, and those of Experiment 1 (which were performed using the same test doses), no evidence of toxicity was observed, as would normally be indicated by a thinning of the background bacterial lawn. Experiment 2 treatments in the absence of S9 were performed up to 5000 µg/plate, and following toxicity resulting in repeat treatments, up to estimates of the lower limit of toxicity in the presence of S9. Experiment 2 treatments were all performed using narrowed dose ranges, in order to more closely investigate those doses of Incozol 2 considered most likely to induce any mutagenic activity. In addition, Experiment 2 treatments in the presence of S9 also employed a pre-incubation step, in order to increase the range of mutagenic chemicals that can be detected using this assay system. No evidence of toxicity was observed following Experiment 2 treatments in the absence of S9. The initial and first repeat treatments of Experiment 2 in the presence of S9 resulted in toxicity at multiple test doses, rendering the data unsuitable for valid mutagenicity assessment. The second repeat treatments produced evidence of toxicity at only the highest one or two test doses, and these results were therefore used as the mutagenicity data for these treatments. 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. Incozol 2 treatments of all the test strains, either in the absence or in the presence of S9, produced no increases in revertant numbers that were sufficient to be considered as evidence of mutagenic activity. It was concluded that Incozol 2 did not induce mutation in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), when tested under the conditions employed in this study, which included treatment at concentrations up to either 5000 µg/plate or the lower limit of the toxic range, both in the absence and in the presence of a rat liver metabolic activation system (S9) (Corning Hazleton, 1996).