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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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

2,2-Dimethyl-1,3-dioxolane-4-methanol was found to be not mutagenic in mouse lymphoma L5178Y

Link to relevant study records

Reference

Reference 1

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.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

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

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

Target gene:

His+ for S. typhimurium

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

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

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.

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

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

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

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

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'.

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 20th 2020 to June 19th 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y/TK+/--3.7.2C mouse lymphoma cells.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem
GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that have been
dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and
ß-naphthoflavone (100 mg/kg body weight).

Test concentrations with justification for top dose:

125 , 250, 500, 1000 and 1322 µg/mLwith and without S-9

Vehicle / solvent:

The vehicle for the test item was Milli-Q water (Millipore Corp., Bedford, MA., USA).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: cyclophosphamide,

Details on test system and experimental conditions:

no further information

Evaluation criteria:

According to criteria set out in OECD Test Guidelines No 490 and Guidelines for Screening Mutagenicity Testing of Chemicals

Statistics:

Determination of the Mutant Colonies
Calculation of the survival of viability
Calculation of the mutation frequency
Acceptabiloty Criteria

A mutation assay is considered acceptable if it meets the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120%
in order to have an acceptable number of surviving cells analysed for expression of the
TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 106 survivors and
≤ 170 per 106 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls
should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the
24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation
frequency, that is, an increase above the spontaneous background MF (an induced MF
(IMF)) of at least 300 x 10-6. At least 40% of the IMF should be reflected in the small
colony MF. And/or, the positive control has an increase in the small colony MF of at least
150 x 10-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10 -6

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Under the test conditions of this study, no biologically relevant increase in the mutation frequency at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix.

Executive summary:

In in vitro mammalian cell gene mutation test, performed according to the OECD 490 TG guideline and in compliance with GLP, L5178Y/TK+/--3.7.2C mouse lymphoma cells ) were exposed to 2,2-Dimethyl-1,3-dioxolane-4-methanol diluted in water at the following concentrations 125 , 250, 500, 1000 and 1322 µg/mL in the presence or in the absence of metabolic activation.

No significant toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix in both experiments.

No biologically relevant increase in the mutation frequency at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix.

Under the test conditions of this study, the test substance is not considered as mutagenic in mammalian cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

2,2-Dimethyl-1,3-dioxolane-4-methanol did not induce any increase in the number of micronucleated polychromatic erythrocytes in the bone marrow of mice dosed up to 2000 mg/kw bw and is therefore considered non clastogenic.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 15 May 2009 to 30 September 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD Guideline 474 without any deviation.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Remarks:

19 November 2008

Type of assay:

micronucleus assay

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

Species:

mouse

Strain:

Swiss

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Paulistec, Mairiporã - SP
- Weight at study initiation: no data
- Age at study initiation: 9 weeks
- Assigned to test groups randomly: no data
- Housing: Animals were housed in polypropylene cages measuring 30 x 20 x 13 cm, with six animals per cage.
- Diet: Pelleted commercial diet (Biobase Biotec, batch R10/09), ad libitum
- Water: Filtered water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 22 °C
- Humidity: 70 %
- Ventilation: no data
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: 19 August 2009 To: 21 August 2009

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: Deionized water
- Concentration of test material in vehicle: no data
- Stability in vehicle: no data

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Test item was dissolved in deionized water.

DOSE VOLUME: 5 mL/kg bw

Duration of treatment / exposure:

- Vehicle control, positive control and treated groups: Two consecutive days treatment at 24 h interval

Frequency of treatment:

- Vehicle control and treated groups: Once daily for two days

Post exposure period:

Vehicle control, positive control and treated groups: from 18 to 24 h after the second treatment

Remarks:

Doses / Concentrations:
Preliminary test assay: : 2000 mg/kg bw/day Main assay: 2000 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

Preliminary toxicity assay: 5 animals (2 in a first assay, 3 others to confirm the observations)

Main assay:
- Negative and positive control group: 6 animals/group
- Treated group-2000 mg/kg bw/day: 6 animals

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: Intraperitoneal route
- Dose: 50 mg/kg bw
- Dose volume: 5 mL/kg bw

Tissues and cell types examined:

- Polychromatic (PCE) and normochromatic (NCE) erythrocyte ratio was established by scoring a total of 1500 erythrocytes (PCE+NCE) per animal.
- For each animal, the micronucleated PCE (MNPCE) were counted in 3000 PCE.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- Dose levels were selected on the basis of results of preliminary toxicity assay in which the test item was administered by intraperitoneal injection to Swiss mice at the dose of 2000 mg/kg bw/day for two consecutive days at 24 h interval.

TREATMENT AND SAMPLING TIMES:
- Two consecutive treatments at 24 h interval followed by sacrificing the animals 18 to 24 h after second treatment (vehicle control, positive control and treatment groups)

DETAILS OF SLIDE PREPARATION:
- At the sampling time, all the animals were sacrificed.
- Femurs of the mice were removed and the bone marrow was extracted with foetal calf serum.
- After centrifugation and re-suspension in a small volume of foetal calf serum, bone marrow smears were prepared in glass slides.
- Slides were air-dried, fixed, stained using Wright-Giemsa and blind evaluated using an optical microscope.

Evaluation criteria:

- Micronuclei are uniform, darkly stained, more or less round bodies in the cytoplasm of erythrocytes. Inclusions which are reflective, improperly shaped or stained, or which are not in the focal plan of the call are judged to be artifacts and are not scored as micronuclei. Cells containing more than one micronucleus are only counted once.
- Positive and negative controls were compared to ensure that the assay was performed according to the prescribed standards.
- A test substance is considered to be active in the test system if there is a clear dose-related increase in the micronuclei frequency compared to negative control at any tested dose.
- Historical negative control data from the laboratory may also be used for comparison between groups.
- The biological relevance of the results was considered together with the statistical significance to evaluate the effects.

Statistics:

- A modified shi-square test according to Pereira (1991) was employed for analysis of the results.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Mild transient ataxia

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF PRELIMINARY TOXICITY ASSAY
- Dose: 2000 mg/kg bw/day for two consecutive days at 24 h interval.
- Mortality: No mortality was observed.
- Clinical signs: All the treated-animals presented mild ataxia after the applications on days 1 and 2 with complete reversion one hour after each injection. The highest dose level which did not produce severe toxicity or lethality was 2000 mg/kg bw. No clinical signs were observed in animals of the negative and positive control groups.

RESULTS OF MAIN ASSAY
Induction of micronuclei (for Micronucleus assay):
- The positive control group treated with cyclophosphamide induced a highly significant increase in the frequency of micronuclei indicating sensitivity of the test system (388 micronuclei were observed in the 18000 PCE).
- In the vehicle control group, the results were consistent to the historical data.
- When animals treated with the test item were compared to the concurrent negative control group, a lower number of micronucleated PCE was observed in the treated group. While 15 micronuclei were observed in the 18000 PCE of the negative control group, 11 micronuclei were observed in the 18000 PCE of the group test with 2000 mg/kg bw of the test item. For this reason, chi-square test was not necessary for comparison between negative control and experimental group.
- See table 7.6.2/1

Ratio of PCE/NCE (for Micronucleus assay):
Six animals were analyzed in the experimental and control groups. A total of 18000 cells were analyzed per group. The quality of the slides allowed a clear differentiation between PCE and NCE. Analysis of the cells showed an approximate 1:1 PCE/NCE rate indicating that there was not a very highly toxic effect of the test substance in the bone marrow of the treated animals

HISTORICAL DATA
- Results were compared with the historical data of the laboratory.

Table 7.6.2/1: Results of micronucleus assay

Dose	PCE/NCE ratio	MNPCE
		Number	%
Vehicle	1.05	2	0.07
	1.14	3	0.10
	1.04	2	0.07
	0.92	2	0.07
	1.15	2	0.07
	1.01	4	0.13
Test item 2000 mg/kg bw/day	1.01	2	0.07
	1.12	2	0.07
	0.99	1	0.03
	1.08	2	0.07
	1.05	2	0.07
	1.12	2	0.07
Cyclophosphamide 50 mg/kg bw	0.83	70	2.33
	0.83	65	2.17
	0.75	66	2.20
	0.75	59	1.97
	0.83	65	2.17
	0.75	63	2.10

 

Conclusions:

Under the test conditions, PEX-01 was not clastogenic and not aneugenic in bone marrow cells of mice.

Executive summary:

In an in vivo bone marrow micronucleus assay performed according to OECD Guideline 474 and in compliance with GLP, Swiss male mice (6/group) were administered 2,2-Dimethyl-1,3-dioxolane-4-methanol in distilled water by intraperitoneal injections at the dose level of 2000 mg/kg bw/day with 2 consecutive treatments at 24 h interval. The vehicle and positive control groups (6 animals/group) received distilled water and cyclophosphamide at 50 mg/kg bw, respectively. Bone marrow smears were obtained from treatment, vehicle and positive control groups approximately 24 h after last administration. Polychromatic (PCE) and normochromatic (NCE) erythrocyte ratio was established by scoring a total of 1500 erythrocytes. For each animal, the micronuclei were counted in 3000 polychromatic erythrocytes. A preliminary toxicity study on two animals was conducted before the main study at the dose of 2000 mg/kg bw/day for two consecutive days and was confirmed on three other animals.

 

When animals treated with the test item were compared to the concurrent negative control group, a lower number of micronucleated PCE was observed in the treated group. For this reason, statistical analysis by chi-square test was not necessary for comparison between negative control and experimental group. Positive control induced a statistically significant increase in micronucleated polychromatic erythrocytes indicating the validity of the study.

 

Under the test conditions, the test substance did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow after two intraperitoneal administrations with a 24 hours interval at 2000 mg/kg bw. 2,2-Dimethyl-1,3-dioxolane-4-methanol is therefore not considered as clastogenic nor as aneugenic in bone marrow cells of mice.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In a GLP-compliant reverse gene mutation assay in bacteria performed according to the OECD Guideline 471, 2,2-Dimethyl-1,3-dioxolane-4-methanol did not induce an increase in the number of revertants in S. typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 with and without metabolic activation. PEX-01 was therefore considered as not mutagenic in these strains.

 

In a GLP-compliantin in vitro mammalian gene mutation test with L5178Y mousse lymphonma cell

was performed according to OECD Guideline 490 and ,2,2-Dimethyl-1,3-dioxolane-4-methanol is concidered as not mutagenic

 

In a GLP-compliant in vivo bone marrow micronucleus assay performed according to OECD Guideline 474, 2,2-Dimethyl-1,3-dioxolane-4-methanol did not induce any increase in the number of micronucleated polychromatic erythrocytes in the bone marrow of Swiss mice dosed up to 2000 mg/kg bw/day for two consecutive days.

Justification for classification or non-classification

As 2,2-Dimethyl-1,3-dioxolane-4-methanol was negative in an in vitro mutation assay in bacteria (OCDE 471) and in vitro mammalian celle gene mutation test ( OCDE 490 TG). it is not classified as mutagenic according to the CLP Regulation (EC) N°1272/2008 and GHS UN.

 

As 2,2-Dimethyl-1,3-dioxolane-4-methanol was not clastogenic in an in vivo bone marrow micronucleus assay, it is not classified as clastogenic according to the CLP Regulation (EC) N°1272/2008 and GHS UN.