Guerbet alcohols, C24-26, branched and cyclic

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:

July to September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21. July 1997

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:

Guerbet alcohols, C24-26, branched and cyclic

EC Number:

942-520-9

Cas Number:

2041102-78-5

Molecular formula:

not applicable, UVCB

IUPAC Name:

Guerbet alcohols, C24-26, branched and cyclic

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Isofol 2426 S
- Substance type: pure active substance
- Physical state: colourless liquid
- Storage condition of test material: room temperature: 20 ± 5 °C

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA97a

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1535

Metabolic activation:

with and without

Metabolic activation system:

S9 from the livers male Sprague-Dawley rats, treated with 500 mg Aroclor 1254/kg bw

Test concentrations with justification for top dose:

First experiment: 5000 /1500 / 500 / 150 / 50 µg/plate
second experiment: 5000 / 2500 / 1250 / 625 / 312 / 156 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: In a preliminary test, the solubility of the test item was determined in demineralised water, DMSO and ethanol. The test item is only soluble in ethanol in a concentration of 50 g/L.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylene Diamine, 20 µg/plate, with strains TA97a, TA98 and TA102; Sodium Azide, 1 µg/plate with strains TA100 and TA1535

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Amino-anthracene, 1 µg/plate, with strains TA97a, TA100, TA102 and TA1535; Benzo-a-pyrene, 20 µg/plyte with strain TA98

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation in the first experiment; preincubation in the second experiment

DURATION
- Preincubation period: 20 min (only in the second experiment)
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY:
- Method:in the main experiments: evaluation of background lawn, reduction in number of revertants in comparison to negativ/solvents control

OTHER EXAMINATIONS:
- Visual counting of mutant colonies, a spreadsheet software (Microsoft Excel) was used to calculate mean values and standard deviations.
- Quality control of bacterial strains: genotype confirmation for each batch of bacteria before stock culture preparation: all bacterial strains were tested for histidine requirement, ampicillin resistence, crystal violet sensitivity, UV sensitivity and spontaneous revertants, furthermore the following examinations were performed: determination of titre, toxicity control, sterility control and positive control

Evaluation criteria:

The colonies were counted visually, the numbers were recorded. A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control. The increase factor f(I) of revertant induction (mean revertants divided by mean spontaneous revertants) and the absolute number of revertants (revertants less mean spontaneous revertants) were also calculated.

A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate in at least one strain exceeding an increase factor of 2 (in tester strains TA 97a, TA98, TA100 and TA102) and an increase factor of 3 (in tester strain TA1535) as compared to the reversion rate of the solvent control can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.

Statistics:

not performed

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA 97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: not soluble in water
- Precipitation: Precipitated/undissolved test item was not observed at any of the concentrations tested.
- Other confounding effects: nothing mentioned

COMPARISON WITH HISTORICAL CONTROL DATA: Nearly all determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory, differences were only marginal and no critical impact on the outcome of the study was expected. All positive control showed mutagenic effects with and without metabolic activation.

Any other information on results incl. tables

Table #1: First Mutation Assay (Direct Plate Incorporation Method)

	TA 97a				TA 98				TA 100
	- S9 mix		+ S9 mix		- S9 mix		+ S9 mix		- S9 mix		+ S9 mix
Dose level[µg/plate]	Mean revertants per plate ± SD	f (I)	Mean revertants per plate ± SD	f (I)	Mean revertants per plate ± SD	f (I)	Mean revertantsper plate± SD	f (I)	Mean revertants per plate ± SD	f (I)	Mean revertants per plate ± SD	f (I)
H2O	106 ± 12.7	-	112 ± 19.6	-	12 ± 4.0	-	14 ± 5.6	-	101 ± 15.1	-	117 ± 10.4	-
DMSO	114 ± 9.0	-	98 ± 10.0	-	11 ± 2.6	-	14 ± 1.7	-	117 ± 5.5	-	115 ± 3.0	-
5000	116 ± 11.4	1.05	121 ± 4.0	1.21	12 ± 2.1	0.86	14 ± 0.6	1.00	113 ± 4.0	0.97	108 ± 12.2	0.95
1500	118 ± 5.0	1.06	121 ± 8.0	1.21	12 ± 2.5	0.86	11 ± 1.2	0.79	113 ± 5.1	0.97	109 ± 13.2	0.96
500	126 ± 1.5	1.14	125 ± 8.5	1.25	10 ± 0.6	0.71	10 ± 1.5	0.71	112 ± 1.5	0.97	118 ± 5.0	1.04
150	118 ± 7.4	1.06	121 ± 6.2	1.21	12 ± 3.2	0.86	14 ± 0.6	1.00	115 ± 9.3	0.99	111 ± 10.4	0.97
50	102 ± 9.0	0.92	130 ± 28.4	1.30	10 ± 0.6	0.71	12 ± 2.1	0.86	103 ± 6.1	0.89	99 ± 9.0	0.87
Positive controls	1107 ± 80.5	9.71	733 ± 95.4	7.48	333 ± 78.9	30.3	53 ± 12.2	3.79	588 ± 143.2	5.82	660 ± 123.2	5.80
Ethanol	111  ± 2.6	-	100 ± 8.7	-	14 ± 0.6	-	14 ± 2.6		116 ± 4.9		114 ± 4.0

f (I) = increase factor of revertant induction (mean revertants divided by mean spontaneous revertants)

Table #1 (continued): First Mutation Assay (Direct Plate Incorporation Method)

	TA 102				TA 1535
	- S9 mix		+ S9 mix		- S9 mix		+ S9 mix
Dose level [µg/plate]	Mean revertants per plate ± SD	f (I)	Mean revertants per plate ± SD	f (I)	Mean revertants per plate ± SD	f (I)	Mean revertants per plate ± SD	f (I)
H2O	265 ± 45.5	-	400 ± 66.6	-	12 ± 1.7	-	12 ± 2.6	-
DMSO	288 ± 67.3	-	247 ± 36.1	-	12 ± 3.1	-	11 ± 2 .6	-
5000	200 ± 14.4	0.77	284 ± 20.0	1.12	13 ± 3.1	1.30	11 ± 1.7	0.92
1500	263 ± 58.3	1.02	268 ± 24.0	1.06	11 ± 0.6	1.10	10 ± 1.2	0.83
500	333 ± 101.5	1.29	283 ± 10.1	1.12	9 ± 1.2	0.90	12 ± 4.4	1.00
150	349 ± 36.1	1.35	253 ± 26.0	1.00	9 ± 0.6	0.9	12 ± 2.5	0.1.00
50	257 ± 92.7	0.99	308 ± 55.0	1.22	11 ± 3.2	1.10	12 ± 2.5	1.00
Positiv controls	1051 ± 179	3.65	1020 ± 179.6	4.13	113 ± 35.1	9.42	280 ± 49.2	25.45
Ethanol	259 ± 24.4	-	253 ± 41.1	-	10 ± 1.5	-	12 ± 3.8	-

f (I) = increase factor of revertant induction (mean revertants divided by mean spontaneous revertants)

Table #2: Second Mutation Assay (Pre-incubation Method)

	TA 97a				TA 98				TA 100
	- S9 mix		+ S9 mix		- S9 mix		+ S9 mix		- S9 mix		+ S9 mix
Dose level [µg/plate]	Mean revertants per plate ± SD	f (I)	Mean revertants per plate ± SD	f (I)	Mean revertants per plate ± SD	f (I)	Mean revertants per plate ± SD	f (I)	Mean revertants per plate ± SD	f (I)	Mean revertants per plate ± SD	f (I)
H2O	105 ± 5.5	-	138 ± 42.0	-	11 ± 1.2	-	15 ± 2.5	-	109 ± 19.7	-	96 ± 3.8	-
DMSO	135 ± 36.4	-	144 ± 25.0	-	10 ± 0.6	-	11 ± 2.0	-	114 ± 14.6	-	110 ± 22.3	-
5000	157 ± 4.6	0.87	193 ± 34.5	0.94	22 ± 5.2	1.62	27 ± 7.6	2.08	95 ± 5.0	1.01	123 ± 6.4	1.12
2500	191 ± 22.3	1.06	240 ± 24.3	1.17	12 ± 1.2	0.92	20 ± 0.0	1.54	108 ± 5.6	1.15	107 ± 10.5	0.97
1250	157 ± 18.9	0.87	193 ± 14.0	0.94	14 ± 1.5	1.08	16 ± 6.5	1.23	102 ± 6.7	1.09	108 ± 23.1	0.98
625	132 ± 33.7	0.73	162 ± 24.3	0.79	17 ± 0.6	1.31	17 ± 1.5	1.31	119 ± 24.3	1.27	105 ± 30.6	0.95
312	139 ± 18.0	0.77	162 ± 29.9	0.79	14 ± 1.2	1.08	11 ± 0.85	1.20	106± 19.3	1.13	105 ± 12.7	0.95
156	196 ± 72.5	1.08	273 ± 75.1	1.33	14 ± 4.6	1.08	15 ± 4.6	1.15	117 ± 12.5	1.24	108 ± 7.0	0.98
Ethanol	181 ± 25.3	-	205 ± 62.1	-	13 ± 1.2	-	13  ± 3.1	-	94 ± 2.0	-	110 ± 12.7	-
Positive controls	639 ± 48.9	4.73	661 ± 108.9	4.59	227 ± 40.5	22.70	48 ± 13.5	4.36	696 ± 30.2	6.39	729 ± 37.0	6.63

f (I) = increase factor of revertant induction (mean revertants divided by mean spontaneous revertants)

Table #3 (continued): Second Mutation Assay (Pre-incubation Method)

	TA 102				TA 1535
	- S9 mix		+ S9 mix		- S9 mix		+ S9 mix
Dose level [µg/plate]	Mean revertants per plate ± SD	f (I)	Mean revertants per plate ± SD	f (I)	Mean revertants per plate ± SD	f (I)	Mean revertants per plate ± SD	f (I)
H2O	284 ± 10.6	-	236 ± 13.9	-	15 ± 1.0	-	11 ± 0.6	-
DMSO	229 ± 53.3	-	271 ± 37.0	-	10 ± 0.6	-	12 ± 1.5	-
5000	285 ± 55.2	0.97	277 ± 40.5	1.19	10 ± 0.0	0.83	10 ± 0.0	0.71
2500	295 ± 26.0	1.00	271 ± 46.9	1.16	13 ± 1.5	1.08	13 ± 1.0	0.93
1250	273 ± 16.7	0.93	229 ± 40.1	0.98	12 ± 1.5	1.00	10 ± 0.0	0.71
625	249 ± 33.5	0.84	237 ± 8.3	1.02	10 ± 1.0	0.83	12 ± 2.1	0.86
312	259 ± 12.9	0.88	313 ± 25.7	1.34	10 ± 2.5	0.83	10 ± 1.5	0.71
156	208 ± 114.8	0.71	272 ± 97.1	1.17	10 ± 2.0	0.83	10 ± 1.5	0.71
Ethanol	295 ± 15.0	-	233 ± 20.5	-	12 ± 3.5	-	14 ± 3.0	-
Positive controls	1065 ± 69.0	4.65	1033 ± 64.8	3.81	384 ± 186.0	25 .6	115 ± 17.0	9.58

f (I) = increase factor of revertant induction (mean revertants divided by mean spontaneous revertants)

Applicant's summary and conclusion

Conclusions:

The test item did not show mutagenic effects towards Salmonella typhimurium, strains TA97a, TA98, TA100, TA102 and TA1535. Therefore, no concentration-effect relationship could be determined. The test item is considered as not mutagenic under the conditions of the test:

Executive summary:

Two valid experiments were performed following OECD 471 and EU B.13/14 under the conditions of GLP.

First Experiment: Five concentrations of the test item, dissolved in Ethanol (ranging from 50 to 5000 μg/plate) were used. Five genetically changed strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 (genetically manipulated) and TA1535) were exposed to the test item both in the presence and in the absence of a metabolic activation system (liver S9-mix of rats induced by Aroclor 1254) for 48 hours, using the plate incorporation method. None of the concentrations caused a significant increase in the number of revertant colonies in the tested strains. The test item did not show any mutagenic effects in the first experiment. The test item showed no precipitates on the plates in all tested concentrations. No signs of toxicity towards the bacteria could be observed. The background lawn was visible and the number of revertant colonies was not reduced. The sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

Second Experiment: To verify the results of the first experiment, a second experiment was performed, using six concentrations of the test item (ranging from 156 to 5000 μg/plate) and a modification in study performance (pre-incubation method). No significant inrease of the number of revertant colonies in the treatments with and without metabolic activation was observed. No concentration-related increase over the tested range was found. In the highest concentration with metabolic activation (5000 µg/plate) the number of revertant coloies of the stzrain TA98 showed an increase (2.08 fold). But this can be seen as uncritical, because the difference is marginal and no concentration-related increase over the tested range was found

The test item showed no precipitates on the plates in all tested concentrations. No signs of toxicity towards the tested strains cold be observed. The background lawn was visible and the number of revertant colonies was not significantly reduced. The sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

Under the conditions of the test, the test item did not show mutagenic effects towards Salmonella typhimurium, strains TA97a, TA98, TA100, TA102 and TA1535. Therefore, no concentration-effect relationship could be determined. The test item is considered as not mutagenic under the conditions of the test.