2-methyl-4-phenyl-1,3-dioxolane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative with S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA with and without metabolic activation

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 - 27 July 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 Jul 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (for S. typhimurium strains)
trp operon (for E. coli strain)

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone

Test concentrations with justification for top dose:

Pre-experiment: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation.

The pre-experiment is reported as Experiment I.

Experiment II: 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation.

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA), methylmethanesulfonate (MMS)

Remarks:

+S9: 2-AA (2.5 µg/plate, TA1535, TA1537, TA98, TA100; 10 µg/plate, WP2 uvrA); -S9: sodium azide (10 µg/plate, TA1535, TA100); 4-NOPD (10 µg/plate, TA98; 50 µg/plate, TA1537); MMS (2 µL/plate, WP2 uvrA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (Experiment I); preincubation (Experiment II)

DURATION
- Preincubation period: 1 h
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiment

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn

Evaluation criteria:

A test substance is considered as mutagenic if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose-dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose-dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

Mean values and standard deviation were calculated.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

exp. 2: -S9 and +S9: starting at 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

exp. 1: +S9: at 5000 µg/plate; exp. 2: -S9 and +S9: starting at 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

exp. 2: -S9 and +S9: starting at 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

exp. 1: -S9 and +S9: at 5000 µg/plate; exp. 2: -S9 and +S9: starting at 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

exp. 2: -S9 and +S9: starting at 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance precipitated in the overlay agar in the test tubes at 5000 µg/plate with S9 mix. Precipitation of the test substance in the overlay agar on the incubated agar plates was observed at 5000 µg/plate with S9 mix. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment is reported as Experiment I.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Reduced background growth was observed at the higher concentrations with and without metabolic activation in Experiment II. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at higher concentrations with and without metabolic activation in both experiments. Please refer to table 3 and 4.

Table 1. Test results of main test 1 (plate incorporation).

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA1535	TA100	WP2 uvrA	TA1537	TA98
-	0 (DMSO)	18 ± 3	150 ± 17	41 ± 6	15 ± 2	22 ± 1
-	0	13 ± 4	150 ± 16	37 ± 7	15 ± 3	30 ± 2
-	3	16 ± 2	150 ± 18	42 ± 5	16 ± 1	29 ± 6
-	10	14 ± 5	139 ± 13	44 ± 7	16 ± 6	27 ± 6
-	33	13 ± 2	116 ± 32	50 ± 11	12 ± 4	30 ± 5
-	100	17 ± 4	155 ± 12	38 ± 3	18 ± 2	28 ± 6
-	333	20 ± 4	133 ± 27	39 ± 4	10 ± 3	26 ± 1
-	1000	21 ± 2	129 ± 21	31 ± 5	13 ± 4	25 ± 4
-	2500	17 ± 5	82 ± 5	23 ± 7	15 ± 2	30 ± 3
-	5000	10 ± 3	64 ± 9	25 ± 5	16 ± 5	18 ± 3
Positive controls, –S9	Name	NaN3	NaN3	MMS	4-NOPD	4-NOPD
	Concentrations (μg/plate)	10	10	2.0 µL	50	10
	Mean No. of colonies/plate (average of 3 ± SD)	957 ± 12	2018 ± 42	819 ± 36	100 ± 18	409 ± 24
+	0 (DMSO)	13 ± 3	138 ± 10	49 ± 2	16 ± 5	30 ± 4
+	0	16 ± 4	160 ± 16	63 ± 17	18 ± 2	33 ± 12
+	3	13 ± 7	121 ± 16	58 ± 15	14 ± 1	32 ± 10
+	10	12 ± 5	140 ± 5	49 ± 7	13 ± 4	35 ± 3
+	33	16 ± 6	135 ± 20	45 ± 8	14 ± 5	42 ± 7
+	100	16 ± 4	142 ± 16	58 ± 8	12 ± 3	40 ± 5
+	333	14 ± 6	125 ± 21	62 ± 8	16 ± 4	41 ± 8
+	1000	15 ± 3	123 ± 6	37 ± 5	13 ± 4	47 ± 10
+	2500	19 ± 7	63 ± 7	28 ± 8	15 ± 4	44 ± 7
+	5000	19 ± 2^P	38 ± 0^P	26 ± 7^P	3 ± 1^P M	30 ± 3^P
Positive controls, +S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	2.5	2.5	10	2.5	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	405 ± 49	3237 ± 49	457 ± 4	144 ± 9	4429 ± 353

NaN3: Sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: Methylmethanesulfonate

2-AA: 2-aminoanthracene

M: Manual count

P: Precipitate

 

Table 2. Test results of main test 2 (preincubation).

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA1535	TA100	WP2 uvrA	TA1537	TA98
-	0 (DMSO)	10 ± 3	146 ± 11	27 ± 6	10 ± 1	21 ± 5
-	0	10 ± 2	188 ± 5	40 ± 5	7 ± 2	22 ± 1
-	10	10 ± 4	154 ± 7	41 ± 7	11 ± 2	22 ± 4
-	33	12 ± 2	150 ± 14	42 ± 2	13 ± 5	25 ± 7
-	100	14 ± 2	145 ± 18	45 ± 11	12 ± 4	24 ± 2
-	333	12 ± 0	137 ± 17	34 ± 1	13 ± 3	29 ± 1
-	1000	15 ± 5	95 ± 15	41 ± 3	13 ± 0	25 ± 4
-	2500	11 ± 4^M R	56 ± 17^M R	12 ± 5^M R	8 ± 4^M R	11 ± 2^M R
-	5000	6 ± 2^M R	16 ± 14^M R	3 ± 2^M R	6 ± 2^M R	0 ± 0^M R
Positive controls, –S9	Name	NaN3	NaN3	MMS	4-NOPD	4-NOPD
	Concentrations (μg/plate)	10	10	2.0 µL	50	10
	Mean No. of colonies/plate (average of 3 ± SD)	977 ± 166	1851 ± 209	475 ± 29	103 ± 3	432 ± 12
+	0 (DMSO)	13 ± 2	146 ± 7	47 ± 8	13 ± 3	33 ± 7
+	0	12 ± 2	139 ± 33	57 ± 7	13 ± 4	31 ± 8
+	10	12 ± 3	133 ± 8	54 ± 3	14 ± 4	31 ± 8
+	33	12 ± 5	131 ± 16	42 ± 7	11 ± 2	38 ± 6
+	100	13 ± 5	142 ± 16	48 ± 4	9 ± 1	32 ± 3
+	333	11 ± 2	111 ± 6	50 ± 14	9 ± 2	29 ± 2
+	1000	12 ± 4	88 ± 17	54 ± 11	12 ± 3	35 ± 6
+	2500	4 ± 2^M R	25 ± 7^M R	27 ± 2^M R	0 ± 0^M R	14 ± 2^M R
+	5000	0 ± 0^M P R	0 ± 0^M P R	18 ± 4^M P R	0 ± 0^M P R	14 ± 3^M P R
Positive controls, +S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	2.5	2.5	10	2.5	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	421 ± 22	4628 ± 190	429 ± 14	181 ± 27	5196 ± 206

NaN3: Sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: Methylmethanesulfonate

2-AA: 2-aminoanthracene

M: Manual count

P: Precipitate

R: Reduced background growth

Table 3. Reduced background growth in Experiment I and II at different concentrations (µg/plate).

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	/	/	2500 - 5000	2500 - 5000
TA 1537	/	/	2500 - 5000	2500 - 5000
TA 98	/	/	2500 - 5000	2500 - 5000
TA 100	/	/	2500 - 5000	2500 - 5000
WP2 uvrA	/	/	2500 - 5000	2500 - 5000

/ = no reduced background

Table 4. Reduction in the number of spontaneous revertants (below the induction factor of 0.5) in Experiment I and II at different concentrations (µg/plate).

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	/	/	/	2500 - 5000
TA 1537	/	5000	/	2500 - 5000
TA 98	/	/	5000	2500 - 5000
TA 100	5000	5000	2500 - 5000	2500 - 5000
WP2 uvrA	/	/	5000	5000

/ = no toxic effects

In experiment II, the number of colonies did not quite reach the lower limit of our historical control data in the negative control of strain WP2 uvrA without metabolic activation. Since this deviation is rather small, this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study.

Conclusions:

Under the conditions of the Ames test the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) tested with and without metabolic activation.

Executive summary:

The test item precipitated in the overlay agar in the test tubes at 5000μg/plate with S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000μg/plate with S9 mix. The undissolved particles had no influence on the data recording. The plates incubated with the test item showed reduced background growth in all strains used with and without S9 mix. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2015). In this study the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) tested with and without metabolic activation.

Justification for classification or non-classification

Based on the available data, there is no indication that the substance induces genetic toxicity in bacteria. The available data on genotoxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008