Fatty acids, C18-(unsaturated) dimers and their monoesters and diesters with 2-ethylhexanol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-08-24 to 2011-10-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD No. 471 and EU method B.13/14 in a GLP certified testing facility.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

other: liquid

Details on test material:

Name: 400160
Batch no.: KL-11-038
Appearance: yellow, clear viscous liquid
Composition: UVCB, 2-Ethylhexyl esters of dimerised fatty acids, C18-unsaturated
Purity: 100% (GPC)
Production date: 24 March 2011
Expiry date: 24 March 2016
Storage: room temperature (20 ± 5 °C)
dissolved in ethanol

Method

Target gene:

Tester strains TA 98 and TA 97a are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Strain TA 102 is sensitive to oxidizing DNA damage based on AT base pairs at the site of the mutation.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA 97a

Additional strain / cell type characteristics:

other: Mutations in hisD6610, uvrB, pKM 101, and rfa

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: Mutations in hisD3052, uvrB, pKM 101, and rfa

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: Mutations in hisG46, uvrB, pKM 101, and rfa

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: Mutations in hisG428, pKM 101, and rfa

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: Mutations in hisG46, uvrB, and rfa

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate (S9 mix)

Test concentrations with justification for top dose:

plate incorporation method: 5002 / 1501 / 500 / 150 / 50 µg/plate
pre-incubation method: 5003 / 2502 / 1251 / 626 / 313 µg/plate

Vehicle / solvent:

Ethanol

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, H2O, ethanol

Controls 2

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylene diamine

Remarks:

concentration: 20 µg/plate; strains: TA 97a, TA 98 and TA 102 in the absence of metabolic activation

Controls 3

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

concentration: 1 µg/plate; strains: TA 100 and TA 1535 in the absence of metabolic activation

Controls 4

Positive controls:

yes

Positive control substance:

other: 2-Amino-anthracene

Remarks:

concentration: 1 µg/plate; strain: TA 97a, TA 100, TA 102, TA 1535; with metabolic activation

Controls 5

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

concentration: 20 µg/plate; strain: TA 98; with metabolic activation

Details on test system and experimental conditions:

Suspensions of bacterial cells are exposed to the test substance in the presence and in the absence of an exogenous metabolic activation system.

Plate incorporation method:
In the plate incorporation method, these suspensions are mixed with an overlay agar and plated immediately onto minimal medium.

Pre-incubation method
In the pre-incubation method, the treatment mixture is incubated and then mixed with an overlay agar before plating onto minimal medium.
For both techniques, after 2 or 3 days of incubation, revertant colonies are counted and compared to the number of spontaneous revertant colonies on solvent control plates.

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 (“Rev. abs.”, mean revertants minus mean spontaneous revertants) were calculated, too.
A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor ≥2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.

Statistics:

The use of statistics was limited to the calculation of means and standard deviations.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA 97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor ≥ 2) in at least one strain can be observed. A concentration related increase over the range tested can also be taken as a sign of mutagenic activity.

Remarks on result:

other: other: plate incorporation method and pre-incubation method

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1:    Mean No. of Revertants – Plate incorporation method

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
H2O	Mean	113	114	10	10	83	103	194	176	12	9
	SD	11	7	2	2	14	9	29	3	1	2
DMSO	Mean	123	113	12	10	88	86	187	160	9	11
	SD	10	14	2	2	10	14	22	7	2	3
Ethanol	Mean	120	113	12	13	110	117	180	228	15	17
	SD	4	4	2	3	4	4	25	17	2	1
Pos.Contr.	Mean	476	510	242	231	510	472	857	795	227	136
	SD	10	32	9	14	31	28	53	108	16	20
	f(I)	3.87	4.51	20.17	23.10	6.14	5.49	4.58	4.97	18.92	12.36
5002 µg/pl.	Mean	101	107	10	11	99	93	197	200	11	11
	SD	10	5	2	1	6	15	31	14	3	1
	f(I)	0.84	0.95	0.83	0.85	0.90	0.79	1.09	0.88	0.73	0.65
1501 µg/pl.	Mean	109	102	11	13	104	86	182	205	10	13
	SD	3	11	3	3	5	11	21	20	3	3
	f(I)	0.91	0.90	0.92	1.00	0.95	0.74	1.01	0.90	0.67	0.76
500 µg/pl.	Mean	101	99	11	13	95	101	214	198	12	12
	SD	8	6	2	2	6	12	11	12	2	2
	f(I)	0.84	0.88	0.92	1.00	0.86	0.86	1.19	0.87	0.80	0.71
150 µg/pl.	Mean	106	117	12	11	99	90	188	194	13	13
	SD	5	7	3	3	5	15	7	6	4	2
	f(I)	0.88	1.04	1.00	0.85	0.90	0.77	1.04	0.85	0.87	0.76
50 µg/pl.	Mean	108	111	10	14	105	100	203	191	12	12
	SD	6	6	2	4	2	10	9	8	2	2
	f(I)	0.90	0.98	0.83	1.08	0.95	0.85	1.13	0.84	0.80	0.71

SD = standard deviation
f(I) = increase factor

Table 2     Mean No. of Revertants – Pre-incubation method

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
H2O	Mean	111	118	14	15	91	104	204	196	15	14
	SD	9	7	3	4	9	10	18	12	1	1
DMSO	Mean	117	122	13	16	108	100	208	200	16	10
	SD	5	6	4	3	14	9	22	21	1	3
Ethanol	Mean	111	111	15	17	102	112	218	195	12	14
	SD	8	3	2	4	11	6	7	20	2	1
Pos.Contr.	Mean	723	677	377	305	530	547	510	519	282	315
	SD	29	92	40	66	56	96	45	87	24	30
	f(I)	6.18	5.55	29.00	19.06	5.82	5.47	2.45	2.60	18.80	31.50
5003 µg/pl.	Mean	117	120	12	12	85	102	202	211	15	14
	SD	7	9	2	5	17	13	26	21	1	3
	f(I)	1.05	1.08	0.80	0.71	0.83	0.91	0.93	1.08	1.25	1.00
2502 µg/pl.	Mean	109	114	12	12	78	79	210	202	14	16
	SD	3	6	3	1	7	10	19	25	3	1
	f(I)	0.98	1.03	0.80	0.71	0.76	0.71	0.96	1.04	1.17	1.14
1251 µg/pl.	Mean	117	117	11	10	84	91	188	235	17	16
	SD	8	9	2	1	21	17	53	21	1	1
	f(I)	1.05	1.05	0.73	0.59	0.82	0.81	0.86	1.21	1.42	1.14
626 µg/pl.	Mean	114	111	11	10	98	100	215	231	17	17
	SD	3	3	2	1	35	8	8	23	1	1
	f(I)	1.03	1.00	0.73	0.59	0.96	0.89	0.99	1.18	1.42	1.21
313 µg/pl.	Mean	121	120	11	10	82	81	206	196	16	17
	SD	4	14	2	4	5	16	18	20	1	2
	f(I)	1.09	1.08	0.73	0.59	0.80	0.72	0.94	1.01	1.33	1.21

SD = standard deviation

f(I) = increase factor

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

It can be stated, that under the test conditions, 400160 is not mutagenic in the bacterial reverse mutation test using Salmonella typhimurium, strains TA 97a, TA 98, TA 100, TA 102, and TA 1535.

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 97, TA 98a, TA 100, TA 102, TA 1535 of Salmonella typhimurium were exposed to 400160 in sterile ethanol at concentrations of 5002 / 1501 / 500 / 150 / 50 µg/plate in the plate incorporation method and at concentrations of 5003 / 2502 / 1251 / 626 / 313 µg/plate in the pre-incubation method. Both test method were conducted in the presence and absence of mammalian metabolic activation.

400160 was tested at limit concentration (5000 µg/plate) for cytotoxicity. No cytotoxicity could be observed. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study was performed according to test guidelines OECD 471 and EU B.13/14 for bacterial reverse gene mutation data in a GLP certified testing facility.