1,3-Dioxolane-4-methanol, 2-methyl-2-(2-methylpropyl)-

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

Study period:

from 25 February 2011 to 05 May 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to OECD guideline and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

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:

[2-methyl-2-(2-methylpropyl)-1,3-dioxolan-4-yl]methanol

EC Number:

692-614-6

Cas Number:

5660-53-7

Molecular formula:

C9H18O3

IUPAC Name:

[2-methyl-2-(2-methylpropyl)-1,3-dioxolan-4-yl]methanol

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): PEX-2 (previous public name of the registered substance)
- Storage condition of test material: at room temperature

Method

Target gene:

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:

S9 from the liver of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

The dose-levels selected for the preliminary (cytotoxicity) test were 50, 150, 500, 1500 and 5000 µg/plate. The preliminary test was conducted on S. typhimurium TA100 in the absence of metabolic activation system.
Based on the results of the cytotoxicity test, the dose selected for the main test were 50, 150, 500, 1500 and 5000 µg/plate) for the five strains in the presence (S9 mix) and in the absence of metabolic activation.

Vehicle / solvent:

Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was chosen as the solvent vehicle due to its known lack of toxicity to the bacteria.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

mitomycin C

other: ICR 191-Acridine

Remarks:

without S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation): For the direct plate incorporation test without metabolic activation, 0.1 mL of test substance and 0.1 mL of a fresh bacterial culture grown overnight were added to 2.0 mL of top agar. For tests with metabolic activation, 0.5 mL of S9 mix was added to the top agar after the addition of test substance and bacteria.

DURATION
- Incubation period: 72 hours at 37°C after solidification of the plates

NUMBER OF REPLICATES: triplicate for the solutions of test substance and the negative control and duplicate for the positive controls.

DETERMINATION OF CYTOTOXICITY
- Method: The evaluation of the cytotoxicity was performed on the basis of the observation of the inhibition of the background bacterial lawn (transparent instead of opaque and rough) and inhibition or reduction in the number of colonies.

OTHER:
Positive controls:
Without S9 mix:
- sodium azide (SA) for strains TA 1535 and TA 100 (5 µg/plate),
- ICR 191-Acridine (ICR) for strain TA 1537 (1 µg/plate),
- 2-Nitrofluorene (2-NF) for strain TA 98 (2 µg/plate),
- Mitomycin C (MMC) for strain TA 102 (0.5 µg/plate).

With S9 mix:
- 2-Aminoanthracene (2-AA) for strains TA98, TA100, TA102, TA 1535 and TA 1537 (2.5 µg/plate).

Evaluation criteria:

A 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls and a clear dose-relationship are considered as a positive result.

Statistics:

To confirm the positive result the analysis of variance of the data set should indicate significant results (pANOVA <0.05) and a clear dose-related increase in the number of revertants should be observed. The analysis of variance indicates statistically significant differences among the average number of revertants in different concentrations. The dose-response effect is evaluated by means of simple linear regression, where the model Revertants (dose) = intercept +slope.dose is adjusted by least-squares method. The regression indicates the probability of the number of revertants observed in the different concentrations by increased (mutagenicity) or decreased (toxicity).

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See table 7.6.1/1

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
To assess the toxicity of the test item to the bacteria, five dose-levels were tested in the TA 100 strain, without S9 mix. Cytotoxicity is characterised by inhibition of the background bacterial lawn (transparent instead of opaque and rough) and inhibition or reduction in the number of colonies.
No background bacterial lawn inhibition was observed at the tested concentrations.


COMPARISON WITH HISTORICAL CONTROL DATA:
The counts recorded on negative vehicle control plates showed appropriate results with all tester strains in the range finding and definitive tests. Appropriate positive control chemicals induced marked increases in revertant colony numbers with all strains, confirming the sensitivity of cultures and activity of S9 mix. The assays were therefore considered valid.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/1:Mean revertant colony counts in the definitive study

	TA 98			TA 100			TA 102			TA 1535			TA 1537
Conc. (µg/plate)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	26	33	no	183	127	no	308	307	no	22	28	no	10	19	no
50	27	37	no	193	131	no	311	311	no	25	25	no	12	16	no
150	33	31	no	185	145	no	315	308	no	27	22	no	8	23	no
500	24	31	no	169	135	no	293	290	no	31	23	no	10	13	no
1500	21	33	no	176	131	no	312	240	no	29	23	no	9	14	no
5000	28	31	no	185	138	no	303	223	yes^#	27	23	no	9	14	no
2-NF	453
2-AA		1792			1610			1222			1771			190
SA				2170						331
MMC							2449
ICR													1267

 

 

*negative control: DMSO (100 µL/plate)

MA: metabolic activation

2-NF: 2-Nitrofluorene

2-AA: 2-Aminoanthracene

SA: Sodium Azide

MMC: Mitomycin C

ICR: ICR 191-Acridine

^#: with metabolic activation only

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Under the condition of this study, the test item 2-isobutyl-2-methyl-1,3-dioxolane-4-methanol did not induce gene mutations by base pair changes or frameshifts in the genome of Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 at the employed range of concentrations both with and without metabolic activation.

Executive summary:

This study was performed to investigate the potential of the test item, 2-isobutyl-2-methyl-1,3-dioxolane-4-methanol, to induce reverse mutation inSalmonella typhimurium. The study was performed according to OECD guideline no. 471 and in compliance with the Principles of Good Laboratory Practice. 

 A preliminary toxicity test was performed to define the dose-levels of 2-isobutyl-2-methyl-1,3-dioxolane-4-methanol to be used for the mutagenicity study. The test item was then tested according to the direct plate incorporation, with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

Five strains of bacteria Salmonella typhimuriumTA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test item (three plates/dose‑level). After 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

The test item was dissolved in DMSO.

 

Since the test item was non-cytotoxic to strain TA100 in the absence of metabolic activation in the preliminary test, the highest dose-level selected for the main experiment was 5000 µg/plate, according to the criteria specified in the international guidelines. The selected treatment-levels were 50, 150, 500, 1500 and 5000 µg/plate for the five strains, in the presence and in the absence of metabolic activation. The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered to be valid. 

No noteworthy toxicity was noted at any dose-levels in the absence or in the presence of metabolic activation except with strain TA102 with S9 mix were toxicity was noted at the dose-level of 5000 µg/plate. The test item did not induce any substantial increase in the number of revertants, either with or without S9 mix, in any of the five strains.

 

In conclusion, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, in the absence or in the presence of a rat metabolising system therefore 2-isobutyl-2-methyl-1,3-dioxolane-4-methanol is not classified according to Annex VI of the Directive 67/548/CEE and according to EU Regulation 1272/2008 (CLP).