2,2-dimethyl-1,3-dioxolan-4-ylmethanol

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 13 May 2009 to 28 October 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in compliance with OECD Guideline 471 with some deviations: only one experiment by direct incorporation method with negative results not confirmed and absence of confirmation not justified.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(only one experiment by direct incorporation method with negative results not confirmed and absence of confirmation not justified)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

19 November 2008

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2-dimethyl-1,3-dioxolan-4-ylmethanol

EC Number:

202-888-7

EC Name:

2,2-dimethyl-1,3-dioxolan-4-ylmethanol

Cas Number:

100-79-8

Molecular formula:

C6H12O3

IUPAC Name:

(2,2-dimethyl-1,3-dioxolan-4-yl)methanol

Test material form:

liquid

Specific details on test material used for the study:

- Name of test material (as cited in study report): PEX-01; 2,2-Dimetil-4-Hidroximetil-1,3-
Dioxolano
- Physical state: Clear liquid
- Analytical purity: 99.5%
- pH of test substance = 6
- Composition of test material, percentage of components: 99.9% 2,2-Dimethyl-1,3-dioxolane-4-methanol; water: 0.02%; Acidity (Acetic acid): 0.0021%
- Lot/batch No.: 081001
- Expiration date of the lot/batch: 01 October 2009
- Stability under test conditions: Stable at room temperature and under normal conditions of use
- Storage condition of test material: at room temperature

Method

Target gene:

His+ for S. typhimurium

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):

Not applicable

Additional strain / cell type characteristics:

other: see table 7.6.1/1

Metabolic activation:

with and without

Metabolic activation system:

5 % S9 mix; S9 fraction prepared from liver homogenates of Sprague-Dawley male rats induced with Aroclor 1254

Test concentrations with justification for top dose:

PRELIMINARY CYTOTOXICITY ASSAY
- Without S9 mix: 8, 40, 200, 1000 and 5000 μg/plate in TA 100 using plate-incorporation method.

MUTAGENICITY ASSAYS
- Experiment without S9 mix (plate-incorporation method): 650, 1080, 1800, 3000 and 5000 μg/plate (TA 1535, TA 1537, TA 98, TA 100 and TA 102)
- Experiment with S9 mix (plate-incorporation method): 650, 1080, 1800, 3000 and 5000 μg/plate (TA 1535, TA 1537, TA 98, TA 100 and TA 102)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: No data

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Sodium azide (5.0 µg/plate for TA 100 and TA 1535); ICR 191-Acridine (1.0 µg/plate for TA 1537); 2-Nitrofluorene (2.0 µg/plate for TA 98), Mitomycin C (0.5 µg/plate for TA 102)

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2.5 µg/plate for TA98, TA100, TA102, TA1535 and TA1537)

Remarks:

with metabolic activation

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: All five strains were obtained from Moltox Inc. (Annapolis, MD, USA).

METHOD OF APPLICATION: Plate incorporation method

DURATION:
- Incubation period for plates: 72 h at 37 °C

NUMBER OF REPLICATIONS:
- Preliminary cytotoxicity assay: no data
- Mutagenicity assays: 3 plates/dose for each test concentration of test substance and negative
vehicle. 2 plates/dose for positive controls

NUMBER OF CELLS EVALUATED: no data

DETERMINATION OF CYTOTOXICITY:
- Method: Cytotoxicity is characterized by inhibition of the background bacterial lawn and inhibition or reduction in the number of colonies..

OTHERS:
- Sterility of top agar, S9 mix, solvent (DMSO), stock solution of test substance and minimal glucose agar plates were tested.
- Solubility: The solubility of the test substance was determined by dissolving the maximum recommended concentration in DMSO. Insolubility can be demonstrated as precipitation in the solution of test substance that is evident to the unaided eye.
- Cell viability test: Fresh cultures for the test were prepared by inoculating bacterial cultures into Erlenmeyer flasks containing 30 mL of Oxoid nutrient broth No 2. The bacterial cultures were incubated in a gyratory incubator for 16 to 18 hours at 37 ± 1ºC to obtain a density of approximately 108 - 109 cells per mL. The number of viable bacteria was determined by plating 100 μL of the culture dilution 10-6 onto nutrient agar plates. After incubation for 24 ± 2 hours at 37 ± 1ºC the number of colonies was recorded.

Evaluation criteria:

- Results were presented as number of revertant colonies per plate and concentration and by the mutation rate, which corresponds to the rate among number of revertants induced by test substance and number of revertants observed in the negative vehicle control. A test substance is considered to be active in the test system if the mutation rates after 72 hours of incubation of strains exposed to the test substance are ≥ 2 for strains TA98, TA100 and TA102 or ≥ 3 for strains TA1535 and TA1537.

- 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 leastsquares method. The regression indicates the probability of the number of revertants observed in the different concentrations be increased (mutagenicity) or decreased (toxicity).

The acceptance criteria of the assay are:
- presence of background lawn in the test plates;
- spontaneous revertant colony numbers of the negative control are in the range reported in the literature (Maron & Ames, 1983) and consistent to the historical control values;
- historical control data of the spontaneous, solvent and positive controls of the period from September 2007 to June 2009 were used to compare the revertant frequencies.
- positive controls show mutagenic activity in all tested strains.

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 dose-response effect is evaluated by means of simple linear regression, where the model Revertants (dose) = intercept + slope.dose is adjusted by leastsquares method.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

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 applicable

True negative controls validity:

not applicable

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 applicable

True negative controls validity:

not applicable

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 applicable

True negative controls validity:

not applicable

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 applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Solubility: No data reported.

COMPARISON WITH HISTORICAL CONTROL DATA:
Compliance with historical control range data..

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Preliminary cytotoxicity assay:
No background bacterial lawn inhibition was observed at the tested concentrations (i.e. at 8, 40, 200, 1000 and 5000 μg/plate) in the absence of metabolic activation in the TA 100 strain.

Mutagenicity assays:
- No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment at any concentration level, neither in the presence nor absence of metabolic activation.
- No tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological significance.
- Mutation rates after 72 hours of incubation were lower than 2 and the analysis of variance of the data set indicated no significant difference (pANOVA>0.05) among the number of revertants, except for strains TA100 without metabolic activation and TA1535 with metabolic activation. In the assay with the strain TA100 (-S9) oscillations were observed among the numbers of revertants at the different tested concentrations. In the assays with TA1535 (+S9) there was a dose-response increase of the number of revertants. Mutation rates with increasing concentrations were below the border of biological significance as no doubling of the revertants was observed.
- No cytotoxicity was observed in any of the strains although it has been tested up to limit concentrations (5000 µg/plate).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/2: Mean revertant frequencies

Strains	Doses (µg/plate)	Mean revertants per plate
		Mutagenicity assay
		-S9		+S9
		Mean	MR	Mean	MR
TA 1535	Solvent control	46	-	35	-
	650	47	1.02	47	1.37
	1080	44	0.96	47	1.36
	1800	47	1.03	43	1.24
	3000	49	1.08	45	1.29
	5000	46	1.01	48	1.38
	PC	2107	46.14	264	7.62
TA 1537	Solvent control	9	-	16	-
	650	11	1.21	20	1.20
	1080	8	0.86	21	1.29
	1800	7	0.79	20	1.22
	3000	12	1.25	18	1.08
	5000	7	0.71	17	1.06
	PC	1876	201.00	196	12.00
TA 98	Solvent control	36	-	41	-
	650	29	0.80	45	1.10
	1080	39	1.08	37	0.90
	1800	30	0.84	36	0.89
	3000	35	0.97	35	0.85
	5000	38	1.06	45	1.10
	PC	728	20.22	2107	51.39
TA 100	Solvent control	193	-	197	-
	650	181	0.94	195	0.99
	1080	183	0.94	189	0.96
	1800	199	1.03	191	0.97
	3000	196	1.01	201	1.02
	5000	185	0.96	192	0.97
	PC	2359	12.20	2338	11.85
TA 102	Solvent control	313	-	315	-
	650	311	0.99	305	0.97
	1080	317	1.01	309	0.98
	1800	303	0.97	308	0.98
	3000	303	0.97	315	1.00
	5000	309	0.99	307	0.97
	PC	1421	4.54	917	2.91

MR: Mutation rate = number of mutants in the treated / number of mutants in the control

PC: positive controls

Applicant's summary and conclusion

Conclusions:

Under these test conditions, PEX-01 was found to be not mutagenic in S. typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 with and without metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) were exposed to 2,2-Dimethyl-1,3-dioxolane-4-methanol diluted in DMSO at the following concentrations for mutagenicity assay: 650, 1080, 1800, 3000 and 5000 μg/plate in the presence or in the absence of metabolic activation. Metabolic activation system used in this study was 5 % S9 mix. S9 fraction was prepared from liver homogenates of Sprague-Dawley male rats induced with Aroclor 1254. Vehicle and positive control groups were also included in this test.

No substantial increase in revertant colony numbers of any of the five tested strains was observed following treatment at any concentration level, neither in the presence nor absence of metabolic activation. No tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological significance. The positive and vehicle controls induced the appropriate responses in the corresponding strains. No cytotoxicity was observed in any of the strains although it has been tested up to limit concentrations required by the OECD Guideline 471 (5000 µg/plate).

 

Under these test conditions, 2,2-Dimethyl-1,3-dioxolane-4-methanol was found to be not mutagenic in S. typhimurium TA98, TA100, TA102, TA1535 and TA1537 with and without metabolic activation.