3-(2,2-dimethyl-3-hydroxypropyl)toluene

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:

2019-20-18 to 2019-10-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated May 30, 2008

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:

3-(2,2-dimethyl-3-hydroxypropyl)toluene

EC Number:

403-140-4

EC Name:

3-(2,2-dimethyl-3-hydroxypropyl)toluene

Cas Number:

103694-68-4

Molecular formula:

C12H18O

IUPAC Name:

2,2-dimethyl-3-(3-methylphenyl)propan-1-ol

Method

Target gene:

Salmonella typhimurium: histidine (his)
Escherichia coli: tryptophan (trp)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Liver homogenate (S9) from Phenobarbital/ß-naphtoflavone pretreated rats (protein concentration: 29.3 mg/mL)
Type and composition of metabolic activation system:
- source of S9 : Phenobarbital/β-naphthoflavone induced rat liver S9 (Lot. No.: 060619K)
- method of preparation of S9 mix: according to Ames et al. (1977); appropriate quantity of S9 supernatant thawed and mixed with S9 cofactor solution (final concentration: approx. 10% (v/v))
- volume of S9 mix in the final culture medium: 500 μL
- quality controls of S9: Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

Test concentrations with justification for top dose:

The test item was tested at the following concentrations:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II:
Strains TA 1535 and WP2 uvrA: 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate
The remaining strains: 3; 10; 33; 100; 333; 1000; and 2500 μg/plate

The top dose for the main experiments was chosen based on the results of the preliminary study. In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment is reported as experiment I. Since toxic effects were observed in experiment I seven concentrations were tested in experiment II. Based on toxicity observed in experiment I the maximum concentration of 5000 μg/plate was reduced to 2500 μg/plate in some strains. The concentration range included two logarithmic decades.

Vehicle / solvent:

- Solvent used: DMSO (purity > 99%)
- Justification for choice of solvent/vehicle: because of its solubility properties and its relative nontoxicity to the bacteria

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine; 2-aminoanthracene, 2-AA

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Pre-experiment for toxicity: in agar (plate incorporation)
- Experiment I: in agar (plate incorporation)
- Experiment II: Preincubation

TREATMENT:
- Preincubation period: 60 minutes
- Exposure duration/duration of treatment: 48 hours

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Method: Number of revertant colonies

Evaluation criteria:

A test item was considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control was observed. A dose dependent increase was considered biologically relevant if the threshold is reached or exceeded at more than one concentration. An increase of revertant colonies equal or above the threshold at only one concentration was judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative and solvent controls such an increase was not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate (experiment I) respectively up to the highest investigated dose (experiment II). No precipitation of the test item was observed in the overlay agar on the incubated agar plates. The plates incubated with the test item showed reduced background growth in experiment I in all strains used with and without S9 mix. In experiment II normal background growth was observed on the incubated agar plates.
Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains in the presence and absence of S9 mix.

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.

Any other information on results incl. tables

Table 1: Summary of Experiment I

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ± SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvRA
Without Activation	DMSO		11 ± 2^B M	14 ± 3	26 ± 8	98 ± 9	55 ± 9
	Untreated		16 ± 3^B M	18 ± 3	35 ± 3	114 ± 9	54 ± 2
	Test item	3 µg	9 ± 2^B M	14 ± 5	35 ± 9	97 ± 4	51 ± 7
		10 µg	11 ± 2^B M	11 ± 3	33 ± 8	114 ± 16	54 ± 11
		33 µg	10 ± 1^B M	14 ± 3	30 ± 5	99 ± 15	58 ± 9
		100 µg	9 ± 2^B M	15 ± 5	29 ± 4	95 ± 9	48 ± 11
		333 µg	10 ± 3^B M	11 ± 2	26 ± 6	100 ± 15	50 ± 17
		1000 µg	10 ± 2^B M R	7 ± 2^R	15 ± 4^R	73 ± 15^R	23 ± 6^R
		2500 µg	3 ± 2^B M R	1 ± 1^R	5 ± 2^M R	3 ± 1^R	14 ± 1^R M
		5000 µg	3 ± 1^B M R	0 ± 0^R	1 ± 1^M R	1 ± 1^R	11 ± 3^R M
	NaN3	10 µg	1167 ± 49^B M			1883 ± 136
	4-NOPD	10 µg			416 ± 6
	4-NOPD	50 µg
	MMS	2.0 µL					914 ± 104
With Activation	DMSO		11 ± 2^B M	18 ± 3	40 ± 3	117 ± 11	58 ± 4
	Untreated		11 ± 2^B M	17 ± 6	40 ± 11	116 ± 11	62 ± 5
	Test item	3 µg	10 ± 3^B M	17 ± 5	41 ± 7	115 ± 7	60 ± 8
		10 µg	10 ± 2^B M	14 ± 7	35 ± 7	108 ± 19	55 ± 5
		33 µg	11 ± 3^B M	15 ± 4	38 ± 6	113 ± 7	54 ± 4
		100 µg	11 ± 2^B M	14 ± 4	43 ± 8	113 ± 7	56 ± 7
		333 µg	12 ± 3^B M	10 ± 3	37 ± 6	109 ± 6	52 ± 7
		1000 µg	9 ± 1^B M R	4 ± 1^R M	15 ± 2^R M	50 ± 17^R	23 ± 4^R
		2500 µg	1 ± 1^B M R	0 ± 1^R M	2 ± 1^M R	3 ± 1^R	11 ± 3^R M
		5000 µg	0 ± 1^B M R	0 ± 0^R M	0 ± 1^M R	0 ± 0^R	5 ± 1^R M
	2-AA	2.5 µg	349 ± 47^B M	434 ± 7	3097 ± 218	3777 ± 163
	2-AA	10 µg					3.22 ± 22

Key to Positive Controls

NaN3: sodium azide

2-AA: 2-aminoanthracene

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

MMS: methyl methane sulfonate

 

Key to Plate Postfix Codes

R: Reduced background growth

B: Extensive bacterial growth

M: Manual count

 

Table 2: Summary of Experiment II

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ± SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvRA
Without Activation	DMSO		9 ± 3	11 ± 4	27 ± 7	88 ± 5	40 ± 6
	Untreated		14 ± 5	16 ± 4	25 ± 10	102 ± 4	44 ± 3
	Test item	3 µg		15 ± 2	25 ± 8	90 ± 8
		10 µg	11 ± 1	17 ± 6	28 ± 0	89 ± 10	41 ± 1
		33 µg	8 ± 2	14 ± 7	23 ± 2	87 ± 10	46 ± 6
		100 µg	10± 2	15 ± 5	19 ± 6	84 ± 20	44 ± 3
		333 µg	12± 5	8 ± 4	21 ± 3	86 ± 12	34 ± 3
		1000 µg	6 ± 1	1 ± 1	2 ± 0	39 ± 6	18 ± 5
		2500 µg	0 ± 1	1 ± 1	0 ± 1	0 ± 0	0 ± 0
		5000 µg	0 ± 0				0 ± 0
	NaN3	10 µg	1106 ± 78			1705 ± 146
	4-NOPD	10 µg			371 ± 7
	4-NOPD	50 µg		70 ± 11
	MMS	2.0 µL					820 ± 19
With Activation	DMSO		10 ± 0	13 ± 3	36 ± 6	95 ± 7	48 ± 5
	Untreated		12 ± 4	19 ± 3	44 ± 3	100 ± 6	59 ± 6
	Test item	3 µg		14 ± 7	33 ± 8	84 ± 9
		10 µg	14 ± 2	14 ± 3	36 ± 6	87 ± 3	54 ± 2
		33 µg	10 ± 1	15 ± 1	35 ± 9	98 ± 12	54 ± 9
		100 µg	14 ± 3	15 ± 3	39 ± 6	99 ± 3	54 ± 8
		333 µg	13 ± 3	17 ± 2	28 ± 2	68 ± 9	49 ± 6
		1000 µg	9 ± 3	1 ± 1	1 ± 1	1 ± 1	31 ± 3
		2500 µg	0 ± 1	0 ± 0	0 ± 1	0 ± 1	2 ± 1
		5000 µg	0 ± 0				0 ± 0
	2-AA	2.5 µg	331 ± 23	314 ± 11		3166 ± 109
	2-AA	10 µg					306 ± 54

Key to Positive Controls

NaN3: sodium azide

2-AA:2-aminoanthracene

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

MMS: methyl methane sulfonate

Applicant's summary and conclusion

Conclusions:

The test substance was not mutagenic to Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA in the presence and absence of a metabolizing system.

Executive summary:

An in vitro reverse mutation assay study (Ames) was conducted under GLP-conditions according to OECD Guideline 471 and EU Method B13/B14 in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. Two separate Experiments were performed, one as a plate incorporation assay (Experiment I) and a second one as a preincubation assay (Experiment II). All tests were conducted in triplicate and concentrations between 3 μg/plate and 5000 μg/plate were tested. Strains were exposed to the test substance with and without metabolic activation with rat liver S9 mix. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. Cytotoxicity was observed in all strains in the presence and absence of S9 mix. 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. Therefore it was concluded that the test substance did not induce gene mutations and it was considered to be non-mutagenic.