3,5-dimethyl-1,2-dioxolane-3,5-diol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-01-11 until 2012-03-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

According to OECD 476. Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Principles of method if other than guideline:

first experiment: 4 hours treatment with and without metabolic activation
second experiment: 24 hours treatment without metabolic activation, 4 hours treatment with metaoblic activation
third experiment: 4 hours treatment with metabolic activation

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3,5-dimethyl-1,2-dioxolane-3,5-diol

Cas Number:

13784-51-5

IUPAC Name:

3,5-dimethyl-1,2-dioxolane-3,5-diol

Constituent 2

Reference substance name:

2,4-Pentanedione, peroxide (CAS 37187-22-7), 30% in solvent mixture

IUPAC Name:

2,4-Pentanedione, peroxide (CAS 37187-22-7), 30% in solvent mixture

Details on test material:

Identity: 2,4-Pentanedione, peroxide
CAS No.: 037187-22-7
Batch-No.: 1103425149
Molecular Weight: 134.131 g/mol

On the day of the experiment (immediately before treatment), the test item was dissolved in deionised water. The solvent was chosen according to its solubility properties and its relative nontoxicity to the cells. The final concentration of deionised water in the culture medium was 0.5 % v/v.

Method

Target gene:

Thymidine Kinase Locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

other: Clone 3.7.2C

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

Experiment I (4 hours treatment):
without S9 mix: 78.1; 156.3; 312.5; 625.o; 937.5; 1250.0 µg/mL
with S9 mix: 312.5; 625.0; 1250.0; 2500.0; 3750.0; 5000.0 µg/mL
Experiment II (24 hours treatment)
without S9 mix: 75.0; 150.0; 300.0; 400.0; 500.0; 600.0 µg/mL
Experiment II (4 hours treatment)
with S9 mix: 300.0; 600.0; 1200.0; 2400.0; 3000.0; 3600.0 µg/mL
Experiment III (4 hours treatment):
with S9 mix: 300.0; 600.0; 1200.0; 2400.0; 3000.0 µg/mL
Following the expression phase of 48 hours the cultures (printed in bold letters) at 78.1 µg/mL without metabolic activation in experiment I and at 300 µg/mL with metabolic activation in experiment II were not continued since a minimum of only four analysable concentrations is required by the guidelines. The cultures at 5000 µg/mL with metabolic activation in experiment I and 500 and 600 µg/mL without metabolic activation in experiment II were not continued due to exceedingly severe cytotoxic effects.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: solubility properties

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours with and without metabolic activation in experiment 1, 24 hours without metaoblic activation in experiment and 4 hours with metabolic activation in experiment 2
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 to 15 days

SELECTION AGENT (mutation assays): RPMI 1640 medium by addition of 5 µg/mL TFT

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1,5 x 10 exp. 6 cells

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10 exp. 6 cells above the
corresponding solvent control or negative control, respectively.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in negative
and/or vehicle con¬trols and the mutation rates of all negative and/or vehicle controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth, and the cloning efficiency 1 is less than 10 % of the vehicle control
unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used
to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and
dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.

Statistics:

Linear regression analysis (least squares) using SYSTAT 11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA)

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (pH 7.39 in the solvent control versus pH 7.27 at 5000 µg/mL)
- Effects of osmolality: No relevant increase (273 in the solvent control versus 308 at 5000 µg/mL)
- Evaporation from medium: Not examined
- Water solubility: Miscible in water
- Precipitation:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:
The pre-experiment was performed in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Test item concentrations between 39.1 µg/mL and 5000 µg/mL were used with respect to the current guidelines.
Toxic effects leading to RSG values below 50% were observed at 625 µg/mL and above in the absence (4 and 24 hours treatment) and at 2500 µg/mL and above in the presence of metabolic activation (4 hours treatment).
The test medium was checked for precipitation or phase separation at the end of the treatment period (4 hours) before the test item was removed. No precipitation or phase separation was observed up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.
Both, pH value and osmolarity were determined in the pre-experiment at the highest concentrations of the test item and in the solvent control without metabolic activation. There was no relevant shift of both parameters.
The dose range of the main experiments was set according to the cytotoxicity data of the test item. In the main experiments the individual concentrations were generally spaced by a factor of 2.0. A narrower spacing was used at higher concentrations to cover the cytotoxic range more closely.

COMPARISON WITH HISTORICAL CONTROL DATA: Complies


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant toxic effects indicated by a relative total growth of less than 50% were observed in the first experiment at 625 µg/mL and above with and at 1250 µg/mL without metabolic activation. In the second experiment cytotoxic effects as described were noted at 600 µg/mL and above with and at 150 µg/mL and above without metabolic activation. In the third experiment cytotoxic effects occurred at 2400 µg/mL and above. The recommended cytotoxic range of approximately 10-20% relative total growth was covered with and without metabolic activation. The data generated in experiment I at 3750 µg/mL with metabolic activation and in experiment II at 3000 and 3600 µg/mL with metabolic activation are not considered valid since the relative total growth fell even short of the 1% limit set by the IWGT exception criteria.

Remarks on result:

other: strain/cell type: in vitro gene mutation assay with L5178Y cells

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary Table

			relative	mutant		relative	mutant
	conc. µg	S9	total	colonies/		total	colonies/
	per mL	mix	growth	10^6cells	threshold	growth	10^6cells	threshold
Column	1	2	3	4	5	6	7	8
Experiment I / 4 h treatment			culture I			culture II
Solv. control with water		-	100.0	93	219	100.0	130	256
Pos. control with MMS	19.5	-	39.7	434	219	21.7	689	256
Test item	78.1	-	culture was not continued^#			culture was not continued^#
Test item	156.3	-	102.9	119	219	38.8	400	256
Test item	312.5	-	90.6	134	219	48.9	292	256
Test item	625.0	-	130.2	118	219	95.9	197	256
Test item	937.5	-	78.2	197	219	76.2	223	256
Test item	1250.0	-	2.1	258	219	3.0	347	256
Solv. control with water		+	100.0	100	226	100.0	117	243
Pos. control with CPA	3.0	+	53.7	245	226	110.0	250	243
Pos. control with CPA	4.5	+	27.8	415	226	53.0	444	243
Test item	312.5	+	66.9	71	226	103.5	142	243
Test item	625.0	+	49.5	157	226	105.0	148	243
Test item	1250.0	+	46.3	119	226	95.0	150	243
Test item	2500.0	+	17.0	165	226	44.3	126	243
Test item	3750.0	+	0.6	231	226	2.0	267	243
Test item	5000.0	+	culture was not continued^##			culture was not continued^##
Experiment II / 24 h treatment			culture I			culture II
Solv. control with water		-	100.0	87	213	100.0	88	214
Pos. control with MMS	13.0	-	19.5	341	213	23.8	477	214
Test item	75.0	-	78.0	116	213	64.9	134	214
Test item	150.0	-	31.9	169	213	44.9	140	214
Test item	300.0	-	15.2	189	213	12.1	182	214
Test item	400.0	-	2.8	132	213	1.7	132	214
Test item	500.0	-	culture was not continued^##			culture was not continued^##
Test item	600.0	-	culture was not continued^##			culture was not continued^##
Experiment II / 4 h treatment			culture I			culture II
Solv. control with water		+	100.0	94	220	100.0	124	250
Pos. control with CPA	3.0	+	35.8	356	220	33.6	691	250
Pos. control with CPA	4.5	+	24.4	469	220	37.9	663	250
Test item	300.0	+	culture was not continued^#			culture was not continued^#
Test item	600.0	+	39.1	125	220	71.8	158	250
Test item	1200.0	+	18.1	125	220	26.7	170	250
Test item	2400.0	+	5.5	126	220	10.4	155	250
Test item	3000.0	+	2.0	74	220	0.5	235	250
Test item	3600.0	+	0.3	47	220	0.4	369	250
Experiment III / 4 h treatment			culture I			culture II
Solvent control with water		+	100.0	201	327	100.0	83	209
Pos. control with CPA	3.0	+	64.2	259	327	126.2	162	209
Pos. control with CPA	4.5	+	43.3	447	327	41.8	365	209
Test item	300.0	+	82.9	138	327	139.6	53	209
Test item	600.0	+	69.2	191	327	98.0	93	209
Test item	1200.0	+	65.6	136	327	78.6	87	209
Test item	2400.0	+	33.1	166	327	41.0	174	209
Test item	3000.0	+	5.6	338	327	15.4	116	209

threshold = number of mutant colonies per 10⁶cells of each solvent control plus 126

#   culture was not continued since a minimum of only four analysable concentrations is required

##   culture was not continued due to exceedingly severe cytotoxic effects

The values printed in bold are judged as invalid, since even the exception criteria of the IWGT are not met (RTG < 1% in at least one of both parallel cultures).

 

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Executive summary:

The study was performed to investigate the potential of 2,4-Pentanedione, peroxide (CAS 37187-22-7), 30% in solvent mixture to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The assay was performed in three independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed in the absence of metabolic activation with a treatment period of 24 hours in the absence and 4 hours in the presence of metabolic activation. As only three analysed concentrations remained within the acceptable cytotoxic range in the second experiment with metabolic activation this experimental part was repeated as experiment three. So, the third experiment was performed with a treatment period of 4 hours solely in the presence of metabolic activation.

The main experiments were evaluated at the following concentrations:

Experiment I

without S9 mix:                      156.3: 312.5; 625.0; 937.5; 1260.0 µg/mL
with S9 mix:                       312.5; 625.0; 1250.0; 2500.0; 3750.0 µg/mL

Experiment II

without S9 mix:                                     75.0; 150.0; 300.0; 400.0 µg/mL
with S9 mix:                     600.0; 1200.0; 2400.0; 3000.0; 3600.0 µg/mL

Experiment III

with S9 mix:                       300.0; 600.0; 1200.0; 2400.0; 3000.0 µg/mL

Relevant toxic effects indicated by a relative total growth of less than 50% were observed in the first experiment at 625 µg/mL and above with and at 1250 µg/mL without metabolic activation. In the second experiment cytotoxic effects as described were noted at 600 µg/mL and above with and at 150 µg/mL and above without metabolic activation. In the third experiment cytotoxic effects occurred at 2400 µg/mL and above. The recommended cytotoxic range of approximately 10-20% relative total growth was covered with and without metabolic activation. The data generated in experiment I at 3750 µg/mL with metabolic activation and in experiment II at 3000 and 3600 µg/mL with metabolic activation are not considered valid since the relative total growth fell even short of the 1% limit set by the IWGT exception criteria.

No substantial and reproducible dose dependent increase of the mutation frequency was observed with and without metabolic activation at acceptable levels of cytotoxicity. The isolated increase of the mutation frequency noted at a severely cytotoxic concentration of 1250 µg/mL in both cultures of the first experiment without metabolic activation was not reproduced in the second experiment without metabolic activation at about the same cytotoxic level generated at 400 µg/mL. Furthermore, this increase was not dose dependent as indicated by the lacking statistical significance in one of the parallel cultures and consequently judged as biologically irrelevant. The increased mutation frequency exceeding the threshold in just one of both parallel cultures (culture II of the first experiment without metabolic activation at 156.3 and 312.5 µg/mL and culture I of the third experiment at 3000 µg/mL) was not considered relevant as the increase was not reproduced in the parallel cultures.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency using SYSTAT^âstatistics software. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely determined in the first culture of the first experiment without metabolic activation. However, this trend was not reproduced in the parallel culture and therefore, not considered biologically relevant as discussed above.

The highest solvent control value (201 colonies per 10⁶cells) exceeded the recommended 50 – 170 x 10⁶control range as stated under paragraph 10.12,acceptability of the assay of this report. The mean value of both parallel cultures however, (201 and 83 equal to 142 colonies per 10⁶cells) is fully acceptable. The cloning efficiency (viability) slightly exceeded the upper limit of 120% (127%) in the solvent control of the first culture of the first experiment with metabolic activation. This minor deviation was judged as irrelevant as it was rather minor and the cloning efficiency of the parallel culture remained within the acceptable range.

MMS (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and CPA (3.0 and 4.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies and an increase of the relative quantity of small versus large induced colonies with at least one of the concentrations.

Under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.