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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test (OECD 471): negative

HPRT Test (OECD 476): negative

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:

2019-20-18 to 2019-10-30

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 July 21, 1997

Deviations:

no

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

Target gene:

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

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

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.

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

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

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

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

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.

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1987-03-26

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

please refer to principles of method if other than guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

yes

Remarks:

please refer to principles of method if other than guideline

Principles of method if other than guideline:

only four strains were tested

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9-Mix

Test concentrations with justification for top dose:

Experiment I
Concentration range in the main test (with metabolic activation): 0.8; 4; 20; 100; 500 µg/plate
Concentration range in the main test (without metabolic activation): 0.8; 4; 20; 100; 500 µg/plate

Experiment II
Concentration range in the main test (with metabolic activation): 1.6; 8; 40; 200; 1000 µg/plate
Concentration range in the main test (without metabolic activation): 1.6; 8; 40; 200; 1000 µg/plate

Vehicle / solvent:

Solvent: DMSO

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene (+S9)

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added: in agar (plate incorporation)

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 applicable

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Table 1 Experiment I, without S9

Concentration (µg/plate)	TA 100 (mean±SD	TA 1535 (mean±SD	TA 98 (mean±SD	TA 1538 (mean±SD	TA 1537 (mean±SD
0	76± 11	11± 2	15±3	Not tested	8± 3
0.8	81± 3	13±5	20± 9	Not tested	6± 2
4.0	88± 6	11± 5	21± 5	Not tested	8± 4
20.0	88± 12	15± 5	18± 4	Not tested	5± 3
100.0	85± 12	12± 3	19± 8	Not tested	7± 5
500.0	78± 9	11± 2	6± 2	Not tested	3±2
Na-azide	739± 58	725± 55		Not tested
2-Nitrofluorene			519± 105	Not tested
9-Aminoacridine				Not tested	554±111

 

Table 2 Experiment I, with S9

Concentration (µg/plate)	TA 100 (mean +/- SD	TA 1535 (mean +/- SD	TA 98 (mean +/- SD	TA 1538 (mean +/- SD	TA 1537 (mean +/- SD
0	92± 9	25± 7	21± 5	Not tested	10± 6
0.8	97± 6	24± 7	33± 11	Not tested	7± 2
4.0	102± 12	25± 3	27± 5	Not tested	7± 3
20.0	78± 17	24± 2	28± 7	Not tested	11± 3
100.0	97± 7	32± 6	25± 4	Not tested	7± 2
500.0	78± 10	25± 3	27± 4	Not tested	10± 2
2-aminoanthracene	257± 34	261± 24	121± 10	Not tested	271± 45

 

Table 3 Experiment II, without S9

Concentration (µg/plate)	TA 100 (mean +/- SD	TA 1535 (mean +/- SD	TA 98 (mean +/- SD	TA 1538 (mean +/- SD	TA 1537 (mean +/- SD
0.0	83± 3	27± 4	18± 3	Not tested	7± 2
1.60	76±11	27± 3	16± 3	Not tested	8± 3
8.0	77± 2	28± 6	10± 4	Not tested	10± 1
40.0	94± 3	36± 2	19± 5	Not tested	5± 4
200.0	85± 10	29± 13	13± 4	Not tested	6± 2
1000.0	0± 0	0± 0	0± 0	Not tested	0± 0
Na-azide	836± 113			Not tested
2-Nitrofluorene		753± 107		Not tested
9-Aminoacridine			599± 81	Not tested	303± 48

 

Table 4 Experiment II, with S9

Concentration (µg/plate)	TA 100 (mean +/- SD	TA 1535 (mean +/- SD	TA 98 (mean +/- SD	TA 1538 (mean +/- SD	TA 1537 (mean +/- SD
0.0	85± 5	24± 7	35± 6	Not tested	10± 2
1.60	75± 9	12± 2	30± 11	Not tested	11± 4
8.0	75± 3	14± 4	28± 3	Not tested	8± 3
40.0	80± 2	19± 4	31± 3	Not tested	10± 3
200.0	70± 17	15±1	31± 4	Not tested	13± 6
1000.0	0± 0	0± 0	0± 0	Not tested	0± 0
2-aminoanthracene	206± 19	195± 8	134± 27	Not tested	247± 29

 

Conclusions:

Based on the results of this bacterial reverse mutation assay according to OECD 471 the test item is not considered to be mutagenic.

Executive summary:

A bacterial reverse mutation assay according to OECD 471 (Ames Test) was conducted with the test item in the four Salmonella Typhimurium strains TA 98, TA 100, TA 1535, TA 1537 with and without metabolic activation. Two independent experiments were conducted as plate incorporation test and each concentration was run in triplicates. The revertant number in the test item treated cultures was not increased when compared to the vehicle control. Thus, under the conditions of this study the test item is considered to not be mutagenic in bacteria.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 October 2019 to 07 November 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Japanese Guidelines: "Kanpoan No. 287 - Environment Protection Agency“ “Eisei No. 127 - Ministry of Health & Welfare", "Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry".

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

- Batch No.: 80100030

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate and a good cloning efficiency of untreated cells (as a rule more than 50%) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.
- Normal cell cycle time: doubling time 12 - 16 h in stock cultures

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Thawed stock cultures were propagated at 37 °C in 75 cm^2 plastic flasks. About 2-3×10^6 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%). The cells were sub-cultured once or twice weekly.
All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Phenobarbital/β-naphthoflavone induced rat liver S9
- method of preparation of S9 mix: Phenobarbital/β-naphthoflavone induced rat liver S9 was used as metabolic activation system. An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
The protein concentration of the S9 preparation was 30.7 mg/mL (Lot. No.: 310119) in the pre-experiment and the main experiment.
- concentration or volume of S9 mix and S9 in the final culture medium: 50 µL/mL
- 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:

Pre-test: 13.9, 27.9, 55.7, 111.4, 222.9, 445.8, 891.5, 1783.0 µg/mL
Main-test: 12.5; 25.0; 50.0; 100.0; 125.0*, 150.0*, 200.0*, 400.0* μg/mL
* Data not taken into consideration for mutation analysis due to strong cytotoxic effects (rel. adjusted cloning efficiency I below 10%)

Vehicle / solvent:

- Vehicle used: DMSO; The final concentration of DMSO in the culture medium was 0.5% (v/v).
- Justification for choice of solvent: The solvent was chosen to its solubility properties and as it’s tolerated by the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 5
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 0.7 to 1.2 x 10^7 cells per flask
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 24 hours
- Exposure duration/duration of treatment: 4 hours

FOR GENE MUTATION:
- Expression time: approx. 7 days
- Selection time: 8-11 days
- Fixation time: The colonies used to determine the cloning efficiency were fixed and stained 6 to 8 days after treatment.
- Selective agent used: 6-thioguanine, 11 μg/mL, 4 hours of exposure
- Number of cells seeded: At least 2.0×10^6 cells per experimental point (concentration series plus controls) were subcultivated in 175 cm^2 flasks containing 30 mL medium .
- Method to enumerate mutated colonies: After 8 – 11 days the colonies were stained with 10% methylene blue in 0.01% KOH solution. Colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative survival (RS)

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
The gene mutation assay is considered acceptable if it meets the following criteria:
a) The mean values of the numbers of mutant colonies per 10^6 cells found in the solvent controls of both parallel cultures remain within the 95% confidence interval of the laboratory historical control data range.
b) Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistical significant increase compared with the concurrent solvent control.
c) Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
d) An adequate number of cells and concentrations (at least four test item concentrations) are analysable even for the cultures treated at concentrations that cause 90% cytotoxicity during treatment.
e) The criteria for the selection of the top concentration are fulfilled.

INTERPRETATION OF RESULTS
A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).

Statistics:

A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies (mean values) obtained for the groups treated with the test item was compared to the solvent control groups. A trend was judged as significant whenever the p-value (probability value) was below 0.05. However, both, biological and statistical significance were considered together.

A t-Test was not performed since all mean mutant frequencies were well within the 95% confidence interval of our laboratory’s historical negative control data.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: no relevant shift
- Data on osmolality: no relevant shift
- Precipitation and time of the determination: Phase separation occurred at 445.8 μg/mL and above after 4 hours treatment with and without metabolic activation.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : see tables under section " Any other information on results incl. tables" below
For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : The linear regression analysis showed no significant dose dependent trend of the mutation frequency at any of the experimental groups.
- Statistical analysis; p-value if any : without S9 mix p = 0.294; with S9 mix p= 0.316

HISTORICAL CONTROL DATA
- Positive historical control data:
Positive control EMS (150 and 300 μg/mL, without metabolic activation): Range: 53.9 – 872.3; mean value: 223.3, standard deviation: 101.1; 95% confidence interval: -; number of studies: 199
Positive control DMBA (1.1 and 2.3 μg/mL, with metabolic activation): Range: 55.6 – 739.9; mean value: 188.0, standard deviation: 98.6; 95% confidence interval: -; number of studies: 193
- Solvent control (medium, acetone, water, DMSO, ethanol, THF, EGDE; without metabolic activation): Range: 3.4 – 41.0; mean value: 16.9, standard deviation: 7.0; 95% confidence interval: 2.8 – 30.9; number of studies: 199

- Solvent control (medium, acetone, water, DMSO, ethanol, THF, EGDE; with metabolic activation): Range:2.4 – 40.4; mean value: 16.9, standard deviation: 6.9; 95% confidence interval: 3.1 – 30.7; number of studies: 193

Table: Summary of Results

				relative	relative	re. Adjusted	mutant	95%
	conc.		S9	cloning	cell	cloning	colonies	confidence
	µg/mL	PS	mix	efficiency I	density	efficiency I	106 cells	interval
				%	%	%
Column	1	2	3	4	5	6	7	8
Main Experiment / 4 h treatment				mean values of culture I and II
Solvent control with DMSO			-	100.0	100.0	100.0	16.0	2.8-30.9
Positive control (EMS)	300.0		-	93.2	116.9	108.9	149.9	2.8-30.9
Test item	12.4		-	94.4	86.1	80.5	8.5	2.8-30.9
Test item	25.0		-	88.4	98.7	88.0	9.6	2.8-30.9
Test item	50.0		-	90.1	99.2	89.5	5.4	2.8-30.9
Test item	100.0		-	73.8	97.0	71.6	9.4	2.8-30.9
Test item	125.0		-	3.1	61.4	1.9	7.1	2.8-30.9
Test item	150.0		-	#	3.7	culture was not continued #
Test item	200.0		-	#	1.8	culture was not continued #
Test item	400.0	PS	-	#	3.1	culture was not continued #
Solvent control with DMSO			+	100.0	100.0	100.0	12.4	3.1-30.7
Positive control (DMBA)	2.3		+	98.4	75.5	74.2	347.1	3.1-30.7
Test item	12.5		+	101.2	101.1	102.2	16.2	3.1-30.7
Test item	25.0		+	98.5	76.0	74.9	8.4	3.1-30.7
Test item	50.0		+	93.1	74.1	69.0	11.3	3.1-30.7
Test item	100.0		+	75.0	54.8	40.9	10.0	3.1-30.7
Test item	125.0		+	18.3	15.4	2.8	9.9	3.1-30.7
Test item	150.0		+	#	10.3	culture was not continued #
Test item	200.0		+	#	2.2	culture was not continued #
Test item	400.0	PS	+	#	1.1	culture was not continued #

PS = Phase separation at the beginning and at the end of treatment

# culture was not continued due to exceedingly severe cytotoxic effects

* Data printed in bold were not taken into consideration for mutation analysis due to a rel.adjusted CE I below 10%.

 

Conclusions:

An in vitro gene mutation study (HPRT) was conducted in V79 cells of the Chinese hamster. It was concluded that the test substance did not induce gene mutations at the HPRT locus and therefore was considered to be non-mutagenic.

Executive summary:

An in vitro gene mutation study (HPRT) according to OECD Guideline 476 was conducted in V79 cells of the Chinese hamster. A pre-experiment was performed with and without metabolic activation for the selection of the test substance concentration. Concentrations between 12.5 and 400.0 μg/mL were tested. For the main experiments a concentration range between 12.5 and 400.0 μg/mL was used. However, a relevant cytotoxic effect indicated by the mean relative adjusted cloning efficiency I below 50% was observed in the presence of metabolic activation at 100.0 μg/mL and above and at 125 μg/mL in the absence of metabolic activation. At the concentration of 125.0 μg/mL the mean relative adjusted cloning efficiency I was far below 10% in the presence and absence of metabolic activation. Consequently, the data at this concentration were not taken into consideration for mutation analysis. The main assay was performed with five replicates for each test concentration and the controls. The cells were exposed to the test item for 4 hours with and without metabolic activation. In the main experiment with and without S9 mix the range of the solvent controls was from 16.1 up to 21.6 mutants per 10⁶ cells and the range of the groups treated with the test item was from 3.8 up to 14.0 mutants per 10⁶ cells. No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment. It was concluded that the test substance did not induce gene mutations at the HPRT locus and therefore was considered to be non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo micronucleus test (OECD 474): negative

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987-08-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: adult, 3 months old
- Weight at study initiation: males: 28 - 34 g; females: 26 - 32 g
- Assigned to test groups randomly: yes, under following basis: by lot
- Fasting period before study: no
- Housing: collective caging
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 +/- 2°C
- Humidity: 50 - 80 %
- Air changes: not specified
- Photoperiod: 12 / 12 hrs dark / hrs light

Route of administration:

intraperitoneal

Vehicle:

- Vehicle/solvent used: isotonic saline
- Justification for choice of solvent/vehicle: recommended by guideline

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test solution was prepared by diluting appropriate amounts with isotonic saline.

Duration of treatment / exposure:

single intraperitoneal application

Frequency of treatment:

1

Post exposure period:

24, 48, 72 hours, respectively

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Male: 1000 mg/kg bw; No. of animals: 5; Sacrifice time: 24 hours
Male: 1000 mg/kg bw; No. of animals: 5; Sacrifice time: 48 hours
Male: 1000 mg/kg bw; No. of animals: 5; Sacrifice time: 72 hours

Female: 1000 mg/kg bw; No. of animals: 5; Sacrifice times: 24 hours
Female: 1000 mg/kg bw; No. of animals: 5; Sacrifice times: 48 hours
Female: 1000 mg/kg bw; No. of animals: 5; Sacrifice times: 72 hours

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control: recommended by the guideline
- Route of administration: intraperitoneal
- Doses / concentrations: 40 mg/ kg bw
- Positive control animals were evaluated 24 h after application

Tissues and cell types examined:

Erythrocytes from femura bone marrow.

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION: Preparations were dried, fixed in absolute (99 %) methanol for 5 min. and then allowed to air dry. The slides were stained using a May-Grünwald and Giemsa solution.

METHOD OF ANALYSIS: From each animal 2 preparations were made. Prior to analysis all the slides were randomized and coded (blind evaluation). The cells were examined at a magnification of 1000 x.
A total of 1000 polychromatic erythrocytes was scored from each slide and the number of micronucleated cells in each sample was recorded. The ratio of polychromatic erythrocytes to normochromatic (mature) erythrocytes was calculated on the base of 1000 cells.

Evaluation criteria:

Cell counts are based on a total of 1000 cells per anirnal. Due to own laboratory historical data an incidence of (up to) 0.8 %, respectively of micronucleated polychromatic erythrocytes is considered to be within normal limits.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

clinical signs of toxicity were seen at 1000 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No lethal effects, no cytytoxicity (PCE/NCE).

Dose-Range-Finding

A preliminary dose-range-finding study was performed applying single doses of 5000, 2500 and 1000 mg/kg body weight, respectively by intraperitoneal injection in 2 mice each. Animals treated at 5000 mg/kg body weight survived, but exhibited signs like marked sedation, ventral position, ataxia, cyanosis and writhing reflex up to six hours p.a. A dose level of 2500 mg/kg body weight produced similar signs less severe and 1000 mg/kg produced only slight, individually barely perceptible reactions. This dose level was therefore considered to be a suitable dosage, representing the area of the MTD (maximally tolerated dosage).

 

Number of polychromatic erythrocytes with micronuclei

The number of polychromtic erythrocytes with micronuclei was significantly increased 24 h post injection in the positive control animals. This result confirms the sensitivity of the used animal strain. There was no significant difference to control values at any time point in the males of the test compound treated group. All group mean values lay within normal ranges.

 

Number of polychromatic erythrocvtes

The number of polychromatic erythrocytes was not significantly different in the test item treated animals. It decreased, however, significantly in the positive control group (sum of both sexes).

 

Number of normochromatic erythrocytes

There was an analogue increase in the number of normochromatic erythrocytes in the positive control group, but none in the test item treated mice.

 

Ratio polychromatic normochromatic ervthrocytes

Due to the previous results, the ratio dropped significantly only in the positive control group attaining statistical significance in the sum of both sexes.

 

Conclusions:

The intraperitoneal administration of the test item at a dose level of 1000 mg/kg bw to male and female mice did not produce significant increases in the frequency of micronuclei in the polychromatic erythrocyte stem cells. All mean values lay completely within laboratory own normal ranges. Therefore, under the experimental conditions of this study, the test item is considered to be non-mutagenic at any tested time point in this test system.

Executive summary:

The test item was administered once intraperitoneally at one high dose level of 1000 mg/kg body weight to mice of the NMRI-strain. Samples of the bone marrow were taken after 24 hours, after 48 hours and finally after 72 hours from 5 males and 5 females each. A concurrent control group (C) received the vehicle only and a positive control group (C-pos.) was treated with Cyclophosphamide (EndoxanR) at a dose level of 40 mg/kg body weight. Results from the data analysis show that there was no significant difference in the number of micronucleated polychromatic erythrocytes compared to the control values at any time point. The number of polychromatic and normochromatic erythrocytes and the ratio polychromatic / normochromatic erythrocytes was also not significantly different to the controls in animals treated with the test item at any time point. The positive control group, treated with 40 mg Cyclophosphamide /kg bw revealed a distinct, significant increase in the incidence of micronuclei (62.3‰, p < 0.001). In this group the ratio of polychromatic erythrocytes to normochromatic erythrocytes was significantly decreased in the sum of both sexes in the positive control group, thus indicating signs of toxicity to erythropoiesis. The decrease in the ratio was due to a significant dropping of polychromatic erythrocytes and a significant increase in the number of normochromatic erythrocytes.

The intraperitoneal administration of the test item at a dose level of 1000 mg/kg bw to male and female mice did not produce significant increases in the frequency of micronuclei in the polychromatic erythrocyte stem cells. All mean values lay completely within laboratory own normal ranges. Therefore, under the experimental conditions of this study, the test item is considered to be non-mutagenic at any tested time point in this test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames Test, OECD 471

Key information:

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 colistrain WP2 uvrA (2019). 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 observedin 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.

Supporting information:

The result of above mentioned key study is supported by an additional bacterial reverse mutation assay (1987) according to OECD 471 with the test item in the four Salmonella Typhimurium strains TA 98, TA 100, TA 1535, TA 1537 with and without metabolic activation. Two independent experiments were conducted as plate incorporation test and each concentration was run in triplicates. The revertant number in the test item treated cultures was not increased when compared to the vehicle control. Thus, under the conditions of this study the test item is considered to not be mutagenic in bacteria.

HPRT Test, OECD 476

An in vitro gene mutation study (HPRT) according to OECD Guideline 476 was conducted in V79 cells of the Chinese hamster. A pre-experiment was performed with and without metabolic activation for the selection of the test substance concentration. Concentrations between 12.5 and 400.0 μg/mL were tested. For the main experiments a concentration range between 12.5 and 400.0 μg/mL was used. However, a relevant cytotoxic effect indicated by the mean relative adjusted cloning efficiency I below 50% was observed in the presence of metabolic activation at 100.0 μg/mL and above and at 125 μg/mL in the absence of metabolic activation. At the concentration of 125.0 μg/mL the mean relative adjusted cloning efficiency I was far below 10% in the presence and absence of metabolic activation. Consequently, the data at this concentration were not taken into consideration for mutation analysis. The main assay was performed with five replicates for each test concentration and the controls. The cells were exposed to the test item for 4 hours with and without metabolic activation. In the main experiment with and without S9 mix the range of the solvent controls was from 16.1 up to 21.6 mutants per 10⁶ cells and the range of the groups treated with the test item was from 3.8 up to 14.0 mutants per 10⁶ cells. No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment. It was concluded that the test substance did not induce gene mutations at the HPRT locus and therefore was considered to be non-mutagenic.

In vivo micronucleus test, OECD 474

The intraperitoneal administration of the test item at a dose level of 1000 mg/kg bw to male and female mice did not produce significant increases in the frequency of micronuclei in the polychromatic erythrocyte stem cells. All mean values lay completely within laboratory own normal ranges. Therefore, under the experimental conditions of this study, the test item is considered to be non-mutagenic at any tested time point in this test system.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genotoxicity the test item does not require classification as mutagenic according to Regulation (EC) No 1272/2008 (CLP), as amended for the seventeenth time in Regulation (EU) 2021/849.