2-hexyldecanoic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The Ames test on the target substance 2 -hexyldecanoic acid was negative.

On the target substance no Mouse Lymphoma Mutation study and no Chromosome aberration test is available.

Mouse Lymphoma Mutation study and Chromosome aberration test on the source substance Reaction mass of 2 -butylheptanoic acid and 2 -ethylnonanoic acid and 2 -methyldecanoic acid and 2 -propyloctanoic acid were 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:

March - April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliant

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

from July 21, 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate of Compliance with Good Laboratory Practices according to Directives 2004/9/CE and 2004/10/CE, Groupe Interministeriel des Produits Chimiques, Republique Francaise, Certificat n°: 2016/48, dated 2 Nov 2016

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: delivered from Sponsor, BATCH: 05297/MA
- Expiration date of the lot/batch: October 2021
- Purity test date: 27.10.2017


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (15 - 25°C), non hygroscopic
- Stability: stable under storage conditions
- Solubility and stability of the test substance in the solvent/vehicle: soluble in DMSO, test solutions used immediately after preparation, no further details mentioned
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no data

Target gene:

his D, his C, his G, tryp E

Species / strain / cell type:

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

Remarks:

E. coli uvrA-

Metabolic activation:

with and without

Metabolic activation system:

S9 microsome fraction prepared from Sprague Dawley rat liver homogenate, was provided by MOLTOXTM (POB Box 1189 - 157 Industrial Park Dr - Boone, NC 28607 - USA) (S9 Moltox-11101-5-3919 validated on 28.06.2018 – expiry date: 07.02.2020).

Test concentrations with justification for top dose:

50, 150, 500, 1500 and 5000 µg/plate with and without S9 mix, top dose chosen according to guideline

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test item is soluble in DMSO

Untreated negative controls:

yes

Remarks:

absolute negative control containing no test item; negative controls with solvents used to solubilize positive controls (Acetone, DMSO, NaCl 0.15 M)

Negative solvent / vehicle controls:

yes

Remarks:

vehicle used to solubilize test item (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

9-aminoacridine

2-nitrofluorene

sodium azide

other: cis-Platinum (II) Diammine Dichloride, CAS 15663-27-1: 1 µg/plate, E. coli without S9; 2-anthramine, CAS 613-13-8: 2 µg/plate, TA 98, 100, 1535, 1537 with S9 (plate incorporation); 1 µg/plate, TA 98, 100, 1535, 1537, with S9 (pre-incubation)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Salmonella Typhimurium strains: for each strain, 0.1 mL of the bacterial suspension containing 1-9 x10exp9 bacteria/mL) and 0.1 mL (in -aqueous or -oily vehicle / 50 µL (in non-aqueous or non-oily vehicle as ethanol ….) of each dilution of the original solution and 0.5 mL of sterile phosphate buffer were successively added to 2 mL of overlay agar, maintained supercooled at 45° C, containing 10 % (v/v) of a L-Histidine-D-Biotine solution (0.5 mM).

Escherichia coli strain : in a test tube 0.1 mL of the bacterial suspension containing 1-9 x 10exp9 bacteria/mL and ) and 0.1 mL (in -aqueous or -oily vehicle / 50 µL (in non-aqueous or non-oily vehicle as ethanol ….) of each dilution of the original solution and 0.5 mL of phosphate buffer were successively added to 2 mL of overlay agar maintained super cooled in 45° C containing 5% (v/v) of nutrient broth n° 2 to which are added 5 µL of a L-Tryptophane solution at 2 mg/mL.

Plates were incubated at 37° C over a 48-72-hour period. The number of revertant colonies per plate was counted.

Moreover the following controls were carried out:
Negative controls :
o absolute negative control containing no test item corresponding to the spontaneous reversion rate,
o solvent used to solubilize positive controls : Acetone, DMSO, NaCl 0.15 M
Vehicle used to solubilize test item : DMSO
Positive control

Two methodologies can be used: - either a standard plate incorporation method where the protocol is similar to that described above, except that, 500 µL of S9-mix fraction is quickly added, before pouring the mixture onto the plates ; - or the pre-incubation assay where the solution of the test item solution with the test strain, and 500 µL of S9-mix fraction are preincubated with shaking for 30 min., at 37° C prior to mixing with the overlay agar and pouring onto the minimal agar plate.

1st assay:
- without metabolic activation: in agar (plate incorporation),
- with metabolic activation: in agar (plate incorporation)
2nd assay (this method used because the first assay was negative):
- without metbolic activation: in agar (plate incorporation),
- with metabolic activation: pre-incubation

DURATION
- Preincubation period: 30 min (at 37°C, with shaking)
- Exposure duration: 48-72 h

NUMBER OF REPLICATIONS: 3 plates per experimental point

DETERMINATION OF CYTOTOXICITY
- Method: Bacteriostatic activity was tested in all concentrations in strain TA 100 in a preliminary cytotoxicity experiment: percent of survival in the plates treated with test item concentrations compared to negative control colonies was measured. In a test tube, 0.1 mL of the bacterial suspension (1-9 x 10exp3 bacteria/mL) and 0.1 mL (in -aqueous or -oily vehicle / 50 µL (in non-aqueous or non-oily vehicle as ethanol ….) of the stock solution and dilutions, were successively added to 2 mL of top agar at 45°C, containing 10 % (v/v) of a solution of L-Histidine-D-Biotine (2.5 mM). After homogenization, the content of the tube was poured onto a Petri plate (90 mm in diameter) containing minimal agar (20 mL). 3 plates per concentration were incubated for 48-72 hours at 37°C, and the colonies counted. A negative control containing the blank alone was run in parallel.

In case bacteriostatic activity is detected, the highest concentration to be retained is that exhibiting a bacteriostatic activity of 75 % or less. The precipitate, if present, should not interfere with the scoring. The following four dilutions studied are distributed according to a semi-logarithmic progression.

Evaluation criteria:

Study was judged to be valid if the following criteria were met:
- the bacteriostatic activity of the highest concentration tested is equal of less than 75%,
- the spontaneous reversion rate of the absolute negative control complies with the historical values of the laboratory,
- the spontaneous reversion rate of the solvent is not statistically different from absolute negative control,
- the mean number of revertant colonies obtained for each strain and the corresponding positive control, with and/or without metabolic activation complies with the historical values of the laboratory,
- negative and positive values don't show significant difference with the historical values of the laboratory (± 2 standard deviations).

Criteria for mutagenic activity in this assay:
The result of the test is considered as negative, if the revertant number is below three fold the number of spontaneous reversions, for TA 1535 and TA 1537 strains, and below two fold the number of spontaneous reversions for TA 98, TA 100 and Escherichia coli WP2 (uvrA-)(pKM101) strains without and with metabolic activation.
The result of the test is considered positive if a dependent relationship concentration is obtained in one, or several of the 5 strains, without and/or with metabolic activation, a mutagenic effect being taken into account for a given dilution of test item if the number of revertant colonies is at least two fold that of spontaneous revertant colonies for TA 98, TA 100 and Escherichia coli WP2 (uvrA-)(pKM101), and three fold for TA 1535 and TA 1537.
All results must be confirmed in an independent experiment.

Statistics:

Mean values of replicates with standard deviations were calculated per plate.

Key result

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

Key result

Species / strain:

S. typhimurium TA 1537

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

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

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

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

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:

RANGE-FINDING/SCREENING STUDIES: Results of the bacteriostatic activity control testing show that neither original solution nor dilutions have bacteriostatic effect.

HISTORICAL CONTROL DATA (with number of experiments, ranges, means and standard deviation) (2009-2018)

POSITIVE HISTORICAL CONTROL DATA
TA 1535:
- without metabolic activation, plate incorporation, Sodium Azide: n=1017, range: 190 - 1487, mean: 757.7 ± 217.3
- with metabolic activation, plate incorporation, 2-Anthramine: n=540, range: 26 - 269, mean: 1112.9 ± 56.3
- with metabolic activation, pre-incubation, 2-Anthramine: n=534, range: 25 - 997, mean: 83.0 ± 77.7
TA 1537:
- without metabolic activation, plate incorporation, 9-Aminoacridine: n=1017, range: 224 - 1979, mean: 917.5 ± 465.2
- with metabolic activation, plate incorporation, 2-Anthramine: n=540, range: 24 - 170, mean: 55.4 ± 25.0
- with metabolic activation, pre-incubation, 2-Anthramine: n=534, range: 20 - 182, mean: 47.9 ± 25.3
TA 98:
- without metabolic activation, plate incorporation, 2-Nitrofluorene: n=1017, range: 187 - 1667, mean: 501.5 ± 219.9
- with metabolic activation, plate incorporation, 2-Anthramine: n=540, range: 220 - 1499, mean: 585.0 ± 225.6
- with metabolic activation, pre-incubation, 2-Anthramine: n=534, range: 174 - 1368, mean: 468.0 ± 207.4
TA 100:
- without metabolic activation, plate incorporation, Sodium Azide: n=1017, range: 381 - 1690, mean: 1038.8 ± 331.9
- with metabolic activation, plate incorporation, 2-Anthramine: n=537, range: 361 - 2163, mean: 850.7 ± 373.0
- with metabolic activation, pre-incubation, 2-Anthramine: n=537, range: 291 - 1720, mean: 640.5 ± 279.9
Escherichia coli WP2 (pKM101)(uvr A-)
- without metabolic activation, plate incorporation, cis-Platinium (II) Diamine Dichloride: n=819, range: 248 - 1089, mean: 485.4 ± 171.6
- with metabolic activation, plate incorporation, Dimethyl Benzanthracene: n=402, range: 365 - 1680, mean: 680.9 ± 246.8
- with metabolic activation, pre-incubation, Dimethyl Benzanthracene: n=420, range: 281 - 1680, mean: 668.0 ± 225.4

NEGATIVE (SOLVENT/VEHICLE) HISTORICAL CONTROL DATA
TA 1535:
- without metabolic activation, plate incorporation: n=1017, range: 4 - 23, mean: 11.0 ± 3.7
- with metabolic activation, plate incorporation: n=540, range: 3 - 23, mean: 12.3 ± 4.1
- with metabolic activation, pre-incubation: n=534, range: 5 - 25, mean: 12.9 ± 4.2
TA 1537:
- without metabolic activation, plate incorporation: n=1017, range: 1 - 20, mean: 6.1 ± 2.5
- with metabolic activation, plate incorporation: n=540, range: 1 - 24, mean: 8.0 ± 3.6
- with metabolic activation, pre-incubation: n=534, range: 1 - 21, mean: 8.2 ± 3.5
TA 98:
- without metabolic activation, plate incorporation: n=1017, range: 6 - 29, mean: 15.9 ± 3.9
- with metabolic activation, plate incorporation: n=540, range: 11 - 38, mean: 23.1 ± 5.2
- with metabolic activation, pre-incubation: n=534, range: 10 - 36, mean: 23.1 ± 5.4
TA 100:
- without metabolic activation, plate incorporation: n=1017, range: 40 - 121, mean: 60.1 ± 12.2
- with metabolic activation, plate incorporation: n=537, range: 55 - 152, mean: 90.8 ± 17.9
- with metabolic activation, pre-incubation: n=534, range: 51 - 156, mean: 90.5 ± 19.5
Escherichia coli WP2 (pKM101)(uvr A-)
- without metabolic activation, plate incorporation: n=819, range: 41 - 188, mean: 88.8 ± 36.3
- with metabolic activation, plate incorporation: n=420, range: 80 - 264.0, mean: 156.5 ± 34.0
- with metabolic activation, pre-incubation: n=420, range: 69 - 250, mean: 159.2 ± 36.1

Table #1: Assay n° 1, without metabolic activation, plate incorporation
Concentration [µg/plate]	Revertant colonies / plate
	TA 98		TA 100		TA 1535		TA 1537		E. coli WP2 uvrA
	mean±SD	R	mean±SD	R	mean±SD	R	mean±SD	R	mean±SD	R
Negative control	16.00 ± 1.73	-	69.67 ± 4.51	-	9.33 ± 2.52	-	7.67 ± 2.31	-	159.00 ± 12.12	-
Vehicle (DMSO)	17.33 ± 4.62	-	64.67 ± 5.51	-	13.00± 1.73	-	7.67 ± 3.51	-	169.00 ± 12.12	-
50	19.00 ± 0.00	1.10	59.33 ± 5.03	0.92	12.67 ± 2.52	0.97	7.00 ± 2.65	0.91	107.00 ± 8.72	0.70
150	15.33 ± 3.79	0.88	58.00 ± 2.00	0.90	11.67 ± 1.53	0.90	5.33 ± 2.52	0.70	125.00 ± 30.81	0.74
500	18.00 ± 2.00	1.04	54.67 ± 11.93	0.85	13.33 ± 2.08	1.03	4.43 ± 0.58	0.57	135.33 ± 12.50	0.80
1500	18.33 ± 4.16	1.06	58.00 ± 5.57	0.90	7.67 ± 2.89	0.59	6.00 ± 2.00	0.78	148.00 ± 24.58	0.88
5000	14.00 ± 3.61	0.81	57.00 ± 7.55	0.88	6.67 ± 2.08	0.51	5.33 ± 3.21	1.25	128.33 ± 20.03	0.76
Positive control solvent	17.00 ± 5.20	-	60.67 ± 2.08	-	13.33 ± 2.08	-	8.33 ± 0.58	-	136.33 ± 5.51	-
Positive control	833.00 ± 38.00	49.00	1606.33 ± 47.44	26.48	950.67 ± 114.99	71.3	909.00 ± 94.17	109.08	303.33 ± 18.99	2.22

Table #2: Assay n° 1, with metabolic activation (10% S9 mix), plate incorporation
Concentration [µg/plate]	Revertant colonies / plate
	TA 98		TA 100		TA 1535		TA 1537		E. coli WP2 uvrA
	mean±SD	R	mean±SD	R	mean±SD	R	mean±SD	R	mean±SD	R
Negative control	19.33 ± 4.51	-	75.00 ± 8.72	-	14.67 ± 1.51	-	11.00 ± 3.61	-	247.00 ± 9.00	-
Vehicle (DMSO)	19.67 ± 5.13	-	81.67 ± 5.86	-	15.67 ± 2.31	-	10.00 ± 2.00	-	224.67 ± 6.11	-
50	17.33 ± 1.15	0.88	81.33 ± 2.08	1.00	13.33 ± 2.52	0.85	7.33 ± 3.79	0.73	237.00 ± 12.00	1.05
150	28.00 ± 2.65	1.42	79.33 ± 5.51	0.97	9.33 ± 1.53	0.60	9.33 ± 3.06	0.93	226.00 ± 24.58	1.01
500	27.00 ± 5.29	1.37	72.00 ± 5.57	0.88	11.00 ± 5.29	0.70	8.00 ± 2.65	0.80	220.00 ± 16.09	0.98
1500	27.00 ± 5.29	1.37	66.67 ± 13.65	0.82	9.67 ± 3.06	0.62	8.67 ± 4.51	0.87	232.00 ± 27.62	1.03
5000	24.67 ± 4.62	1.25	71.00 ± 6.56	0.87	9.33 ± 6.81	0.60	9.67 ± 3.51	0.97	268.33 ± 17.56	1.19
Positive control solvent	21.00 ± 5.29	-	76.67 ± 11.59	-	17.00 ± 2.00	-	9.00 ± 2.65	-	214.00 ± 1.73	-
Positive control	430.33 ± 90.14	23.45	625.00 ± 118.65	8.15	106.33 ± 27.54	6.25	47.00 ± 12.53	5.22	477.33 ± 21.83	2.23

Table #3: Assay n° 2, without metabolic activation, plate incorporation
Concentration [µg/plate]	Revertant colonies / plate
	TA 98		TA 100		TA 1535		TA 1537		E. coli WP2 uvrA
	mean±SD	R	mean±SD	R	mean±SD	R	mean±SD	R	mean±SD	R
Negative control	12.67 ± 3.79	-	56.67 ± 7.64	-	8.67 ± 4.73	-	4.33 ± 0.58	-	95.00 ± 14.42	-
Vehicle (DMSO)	14.67 ± 5.77	-	67.67 ± 1.53	-	15.00 ± 5.20	-	3.00 ± 2.00	-	93.33 ± 11.93	-
50	16.67 ± 0.58	1.14	49.00 ± 2.65	0.72	11.67 ± 4.04	0.78	3.67 ± 2.31	1.22	78.67 ± 3.21	0.84
150	17.00 ± 1.73	1.73	53.00 ± 7.81	0.78	10.00 ± 1.00	0.67	5.00 ± 4.36	1.67	90.33 ± 5.69	0.97
500	12.33 ± 4.93	0.84	48.33 ± 2.31	0.71	8.33 ± 1.53	0.56	4.33 ± 1.15	1.44	80.00 ± 2.65	0.86
1500	12.00 ± 3.46	0.82	54.67 ± 4.73	0.81	6.67 ± 1.15	0.44	4.67 ± 2.08	1.56	74.00 ± 9.85	0.79
5000	13.00 ± 5.20	0.89	47.00 ± 4.58	0.69	7.67 ± 2.08	0.51	3.67 ± 2.89	1.22	73.00 ± 5.29	0.78
Positive control solvent	10.00 ± 6.08	-	61.00 ± 3.00	-	12.33 ± 4.04	-	2.00 ± 1.73	-	72.33 ± 10.07	-
Positive control	363.67 ± 37.17	36.17	1363.00 ± 145.32	22.34	1265.00 ± 63.93	102.57	631.33 ± 99.73	118.22	302.33 ± 54.45	4.18

Table #4: Assay n° 2, with metabolic activation, pre-incubation
Concentration [µg/plate]	Revertant colonies / plate
	TA 98		TA 100		TA 1535		TA 1537		E. coli WP2 uvrA
	mean±SD	R	mean±SD	R	mean±SD	R	mean±SD	R	mean±SD	R
Negative control	16.67 ± 1.15	-	79.67 ± 6.43	-	17 -33 ± 1.53	-	5.33 ± 3.51	-	134.67 ± 8.33	-
Vehicle (DMSO)	22.67 ± 7.51	-	100.67 ± 10.02	-	11.67 ± 4.16	-	5.33 ± 4.16	-	113.67 ± 12.50	-
50	22.33 ± 9.81	0.99	65.33 ± 4.93	0.65	11.00 ± 2.00	0.94	7.00 ± 1.00	1.31	121.33 ± 6.66	1.07
150	14.00 ± 1.73	0.62	69.67 ± 13.58	0.69	9.00 ± 7.00	0.77	5.00 ± 1.73	0.94	141.33 ± 3.21	1.24
500	23.00 ± 10.39	1.01	78.00 ± 10.82	0.77	7.00 ± 3.00	0.60	7.67 ± 2.08	1.44	137.00 ± 8.72	1.21
1500	23.00 ± 8.66	1.01	79.33 ± 4.93	0.79	10.00 ± 3.61	0.86	4.33 ± 0.58	0.81	135.33 ± 3.06	1.19
5000	12.67 ± 2.89	0.56	57.33 ± 3.79	0.57	5.67 ± 1.53	0.49	4.00 ± 1.00	0.75	142.67 ± 10.41	1.26
Positive control solvent	17.67 ± 8.33	-	73.00 ± 7.81	-	14.33 ± 5.51	-	5.67 ± 2.08	-	142.67 ± 3.06	-
Positive control	247.67 ± 17.90	14.02	329.33 ± 37.81	4.51	41.67 ± 11.59	2.91	28.33 ± 3.51	5.00	340.00 ± 70.00	2.38

SD = Standard deviation; R = Number of revertant colonies in the presence of the test item/ Number of revertant colonies in the absence of the test item

Conclusions:

Doses (5 000, 1 500, 500, 150 and 50 µg/plate) prepared from solutions of the test item 2-Hexyldecanoic acid do not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2(uvrA¯) (pKM 101) without, or with metabolic activation, according to the OECD Guideline n° 471.

Executive summary:

A bacterial reverse mutation test using Salmonella typhimurium his- and Escherichia coli WP2 (uvrA-)(pKM101) was performed according to OECD Guideline n°471 in compliance with Good Laboratory Practices (GLP).

Solutions obtained from the test item have been tested for their capacity to induce reverse mutation in four Salmonella typhimurium strains and one Escherichia coli WP2 (uvrA-)(pKM101) strain. This study was performed in the absence and presence of metabolic activation. Two independent assays were carried out. For assay n°1, various concentrations were put in contact with the strains in the absence and presence of a metabolic activation system (S9 -mix 10% (v/v)). For assay n°2, various concentrations were put in contact with test strains in absence and presence of a metabolic activation system (S9 -mix 10%(v/v)). For the two assays, negative and postive controls were carried out in parallel. Positive controls induced a significant increase in the number of revertant colonies compared to negative controls. There was no siginificant difference between the number of spontaneous reversions, the number of reversions obtained in the prositive controls (without and with metabolic activation), and the mean of corresponding experimental "historical" values obtained in the laboratory. These results validated the two tests. There was no evidence of any increase in the number of revertant colonies in the presence of the various concentrations of the test item (50, 150, 500, 1500, and 5000 µg/plate), without and with metabolic activation in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100, and in Escherichia coli WP2 (uvrA-)(pKM101). The test item was judged to be non-mutagenic in this test system under the described conditions.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

On the basis of all evaluated data, the similarity of 2-hexyldecanoic acid is justified on basis of the physico-chemical properties and toxicological profiles. There is convincing evidence that the source substance can be used for read-across. (Please see Analogue approach).

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see details below

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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

Conclusions:

Interpretation of results:
negative

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

January to March, 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

On the basis of all evaluated data, the similarity of 2-hexyldecanoic acid is justified on basis of the physico-chemical properties and toxicological profiles. There is convincing evidence that the source substance can be used for read-across. (Please see Analogue approach).

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase locus/TK+/-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
Complete culture medium: RPMI 1640 medium supplemented with 10% heat-inactivated horse serum, 100 U/100 µg/mL penicillin/streptomycin, 1mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B
Treatment medium: RPMI medium supplemented with 5% heat-inactivated horse serum (in case of short term exposure) resp. 7.5% heat-inactivated horse serum (in case of long term exposure), 100 U/100 µg/mL penicillin/streptomycin, 1mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B
Selective medium: RPMI 1640 medium supplemented with 20% heat-inactivated horse serum, 100 U/100 µg/mL penicillin/streptomycin, 1mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B, 5 µg/mL trifluorothymidine (TFT)
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

liver S9 microsomal fraction of phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) induced male Wistar rats

Test concentrations with justification for top dose:

Pre-experiment for toxicity: 0.2/1.0/2.6/5.3/8.0 and 10.6 mM for experiment I with and without S9 mix, 0.05/0.1/0.2/0.5/1.0 and 1.5 mM for experiment II, long term exposure, without S9 mix
Experiment I: 0.05/0.1/0.2/0.5/0.7/0.9/1.1, and 1.3 mM without S9 mix, 0.02/0.05/0.1/0.2/0.5/1.0/1.2 and 1.4 mM with S9 mix
Experiment II: 0.002/0.005/0.01/0.02/0.05/0.1/0.2 and 0.5 mM without S9 mix, 0.15/0.3/0.7/0.9/1.1/1.2/1.3 and 1.4 mM with S9 mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: in the absence of metabolic activation: ethyl methanesulphonate (EMS, 200/300 µg/mL) and methyl methanesulphonate (MMS, 8/10µg/mL); in the presence of metabolic activation: benzo[a]pyrene (B[a]P, 2.5 µg/ml)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment 1: 4 h
Experiment 2: 24 h (without metabolic activation) and 4 h (with metabolic activation)
- Expression time (cells in growth medium): 2 days
- Selection time (incubation with the selection agent): 14 days (mutation selection assay)

SELECTION AGENT: trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 1 (test item and positive controls, 2 (negative control)

NUMBER OF CELLS EVALUATED: mutation selection assay: cells from each experimental group were seeded in four 96-well plates at a density of 200 cells/well in 200 µL selective medium; cloning efficiency assay: seeding a statistical number of 1.6 cells per well in two 96-well plates

DETERMINATION OF CYTOTOXICITY
- Method: measuring the colony-forming ability and the growth rate of cultures (relative suspension growth, recorded over 2 days following treatment)

OTHER EXAMINTATIONS
- Colony sizing: ratio of small to large type mutants

Evaluation criteria:

The assay is considered acceptable if it meets the following criteria:
- at least three out of four of the negative and/or solvent controls is in the range 65% - 120%
- the spontaneous mutant frequency in the negative and/or solvent controls is in the range 50-170 mutants per 10E6 cells
- the cell number of the negative/solvents controls should undergo 8-32 fold increase during a 2 day growth period (short-term treatment) or 32-180 fold increase during a 3 day growth period (long-term treatment)
- the clastogenic positive controls (MMS and B[a]P) have to produce an induced mutant frequency (total mutant frequency minus concurrent negative control mutant frequency) of at least 300 mutants per 10E6 cells with at least 40% of the colonies being small colonies or with an induced small colony mutant frequency of at least 150 mutants per 10E6 cells. The relative total growth (RTG) must be greater than 10%.

Criteria for a positive result:
- the induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 10E6 cells
- a dose-dependent increase in mutant frequency is detected.
Additionally, combined with a positive effect in the mutant frequency, an increased occurence of small colonies (>= 40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations. The biological relevance is considered first for the interpretation of results. Statistical methods might be used an aid in evaluation of the test result.

Criteria for a negative result:
a test item is considered negative if the induced mutant frequency is below the GEF and the trend of the test is negative.

Statistics:

Statistical significance of mean mutant frequency was evaluated at the 5% level (p<0.05) by means of the non-parametric Mann-Whitney test.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

for details see tables 1 and 2 below

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Water solubility: no data, the test substance was soluble in cell culture medium after being treated with ultrasound for 3 minutes at 37°C
- Precipitation: no precipitation was noticed
- Other confounding effects: none

COMPARISON WITH HISTORICAL CONTROL DATA: All values found in the test were within the range of the historical laboratory control data

Table 1a: Pre-experiment for Toxicity, without metabolic activation
Test group	Concentration mM	Suspension Growth	Relative Suspension Growth* [%]
Negative control	0	16.5	100.0
	0	16.4
1	0.2	15.5	94.2
2	1.1	7.7	46.8
3	2.6	0.3	1.9
4	5.3	no viable cells	-
5	8.0	no viable cells	-
6	10.6	no viable cells	-
Table 1b: Pre-experiment for Toxicity, with metabolic activation
Test group	Concentration mM	Suspension Growth	Relative Suspension Growth* [%]
Negative control	0	13.4	100.0
	0	13.6
1	0.2	12.2	90.1
2	1.1	9.1	67.7
3	2.6	0.3	1.9
4	5.3	no viable cells	-
5	8.0	no viable cells	-
6	10.6	no viable cells	-
Table 1c: Pre-experiment II for Toxicity, long-term exposure, without metabolic activation
Test group	Concentration mM	Suspension Growth	Relative Suspension Growth* [%]
Negative control	0	35.9	100.0
	0	33.9
1	0.05	32.9	94.3
2	0.1	31.5	90.2
3	0.2	23.0	65.7
4	0.5	6.0	17.2
5	1.0	0.3	0.8
6	1.5	0.1	0.4
* = (Suspension Growth/Suspension Growth of corresponding controls) x100
Table 2a: Experiment I, without metabolic activation
Test group	Concentration	CE	RCE	RTG	MF	IMF	GEF exceeded	colony sizing
	mM	%	%	%	mutants/10^6cells	mutants/10^6cells		% small colonies
Negative control	0	101.2	100.0	100.0	80.1	/	/	15.8
		108.2				/	/	14.8
3	0.05	104.6	99.9	97.9	71.1	-8.8	-	n.d.
4	0.1	112.0	106.9	109.1	66.5	-13.6	-	n.d.
5	0.2	112.0	106.9	97.6	63.8	-16.3	-	n.d.
6	0.5	84.1	80.3	75.2	94.0	13.9	-	n.d.
7	0.7	98.0	93.6	81.1	50.3	-29.8	-	n.d.
8	0.9	95.0	90.7	61.9	89.7	9.6	-	18.3
9	1.1	114.0	108.8	58.2	79.2	-0.9	-	22.2
10	1.3	130.0	124.1	21.8	63.3	-16.8	-	24.1
EMS	300 µg/mL	93.5	89.3	68.9	690.8*	610.7*	+	n.d.
MMS	10 µg/mL	80.5	76.8	59.4	511.6*	431.5*	+	41.9
CE: Cloning efficiency, CE = (-ln ((96 - (mean value of plates)) / 1.6) x 100
RCE: Relative Cloning Efficiency, RCE = (CE of dose group/CE of corresponding controls)x100
RTG: Relative Total Growth, RTG = (RSG x RCE)/100
MF: Mutant Frequency
GEF: Global Evaluation Factor (126): + = GEF exceeded, - = GEF not exceeded
* statistical significant increase in mutant frequency compared to negative controls
Table 2b: Experiment II, without metabolic activation (24 h treatment)
Test group	Concentration	CE	RCE	RTG	MF	IMF	GEF exceeded	colony sizing
	mM	%	%	%	mutants/10^6cells	mutants/10^6cells		% small colonies
Negative control	0	114.0	100.0	100.0	56.7	/	/	6.1
		102.9				/	/	22.5
1	0.002	96.5	89.0	87.1	60.3	3.5	-	n.d.
2	0.005	90.7	83.6	127.2	75.8	10.9	-	n.d.
3	0.01	85.4	78.7	107.5	101.4*	44.7*	-	n.d.
4	0.02	106.4	98.1	92.7	67.2	10.5	-	n.d.
5	0.05	112.0	103.3	112.6	69.6	12.9	-	n.d.
6	0.1	102.9	94.9	74.3	52.1	-4.6	-	15.4
7	0.2	102.9	94.9	72.2	62.0	5.3	-	13.0
8	0.5	101.2	93.4	20.3	67.6	10.9	-	16.3
EMS	200 µg/mL	32.0	29.5	19.4	3673.2*	3616.5*	+	n.d.
MMS	8 µg/mL	38.9	35.9	25.0	1191.8*	1135.1*	+	51.1
CE: Cloning efficiency, CE = (-ln ((96 - (mean value of plates)) / 1.6) x 100
RCE: Relative Cloning Efficiency, RCE = (CE of dose group/CE of corresponding controls)x100
RTG: Relative Total Growth, RTG = (RSG x RCE)/100
MF: Mutant Frequency
GEF: Global Evaluation Factor (126): + = GEF exceeded, - = GEF not exceeded
* statistical significant increase in mutant frequency compared to negative controls
Table 2c: Experiment I, with metabolic activation
Test group	Concentration	CE	RCE	RTG	MF	IMF	GEF exceeded	colony sizing
	mM	%	%	%	mutants/10^6cells	mutants/10^6cells		% small colonies
Negative control	0	90.7	100.0	100.0	81.0	/	/	10.9
		110.7				/	/	20.8
2	0.02	80.5	80.2	90.2	109.8	28.9	-	n.d.
3	0.05	101.2	100.9	110.3	89.1	8.1	-	n.d.
4	0.1	86.6	86.3	99.7	96.4	15.5	-	n.d.
5	0.2	75.9	75.6	77.3	100.1	19.1	-	n.d.
6	0.5	90.7	90.3	92.7	92.1	11.2	-	n.d.
7	1.0	93.5	93.2	73.4	95.9	15.0	-	22.2
8	1.2	93.5	93.2	46.4	83.0	2.0	-	21.8
9	1.4	93.5	93.2	10.1	121.4*	40.5*	-	20.5
B[a]P	2.5 µg/mL	92.1	91.7	57.7	593.8*	512.8*	+	42.9
CE: Cloning efficiency, CE = (-ln ((96 - (mean value of plates)) / 1.6) x 100
RCE: Relative Cloning Efficiency, RCE = (CE of dose group/CE of corresponding controls)x100
RTG: Relative Total Growth, RTG = (RSG x RCE)/100
MF: Mutant Frequency
GEF: Global Evaluation Factor (126): + = GEF exceeded, - = GEF not exceeded
* statistical significant increase in mutant frequency compared to negative controls
Table 2d: Experiment II, with metabolic activation
Test group	Concentration	CE	RCE	RTG	MF	IMF	GEF exceeded	colony sizing
	mM	%	%	%	mutants/10^6cells	mutants/10^6cells		% small colonies
Negative control	0	110.1	100.0	100.0	72.2	/	/	14.3
		95.0				/	/	18.4
5	0.15	90.7	88.4	79.9	75.6	3.5	-	n.d.
6	0.3	95.0	92.6	100.1	78.5	6.3	-	n.d.
7	0.7	125.0	121.9	103.3	68.1	-4.1	-	n.d.
8	0.9	98.0	95.6	81.9	66.7	-5.5	-	n.d.
9	1.1	82.1	89.8	56.2	79.2	7.1	-	n.d.
10	1.2	120.3	117.4	57.4	63.0	-9.2	-	11.1
11	1.3	120.3	117.4	31.8	64.7	-7.5	-	12.7
12	1.4	104.6	102.0	8.7	77.2	5.1	-	17.5
B{a]P	2.5 µg/mL	85.4	83.3	59.2	477.9*	405.7*	+	42.1
CE: Cloning efficiency, CE = (-ln ((96 - (mean value of plates)) / 1.6) x 100
RCE: Relative Cloning Efficiency, RCE = (CE of dose group/CE of corresponding controls)x100
RTG: Relative Total Growth, RTG = (RSG x RCE)/100
MF: Mutant Frequency
GEF: Global Evaluation Factor (126): + = GEF exceeded, - = GEF not exceeded
* statistical significant increase in mutant frequency compared to negative controls

Conclusions:

Interpretation of results:
negative without metabolic activation
negative with metabolic activation

In the described mutagencity test under the experimental conditions reported, the test item Reaction mass of 2-butylheptanoic acid and 2-ethylnonanoic acid and 2-methyldecanoic acid and 2-propyloctanoic acid is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Executive summary:

The test item Reaction mass of 2 -butylheptanoic acid and 2 -ethylnonanoic acid and 2 -methyldecanoic acid and 2 -proyloctanoic acid was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The selection of the concentrations used in the main experiments was based on data from the pre-experiment. In experiment I 1.3 mM (without metabolic activation) and 1.4 mM (with metybolic activation) were selected as the highest concentrations. In experiment II 0.5 mM (without metabolic activation) and 1.4 mM (with metabolic activation ) were selected as the highest concentrations. Experiment I without and with metabolic activation and experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as a 24 h long-term exposure assay.

The test item was investigated at the following concentrations:

Experiment I without metabolic activation: 0.05, 0.1, 0.2, 0.5, 0.7, 0.9, 1.1 and 1.3 mM and with metabolic activation: 0.02, 0.05, 0.1 ,0.2, 0.5, 1.0, 1.2 and 1.4 mM

Experiment II without metabolic activation: 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2 and 0.5 mM and with metabolic activation: 0.15, 0.3, 0.7, 0.9, 1.1, 1.2, 1.3 and 1.4 mM.

No precipitation of the test item was noted in the experiments. Growth inhibition was observed in experiment I and II without and with metabolic activation.

In experiment I without metabolic activation the relative total growth (RTG) was 21.8% for the highest concentration (1.3 mM) evaluated. The highest concentration evaluated with metabolic activation was 1.4 mM with a RTG of 10.1%. In experiment II without metabolic activation the relative total growth was 20.3% for the highest concentration (0.5 mM) evaluated. The two highest concentrations evaluated with metabolic activation were 1.3 and 1.4 mM with a RTG of 31.8% and 8.7% respectively.

In experiment I and II no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). The Global Evaluation Factor (GEF, defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories) was not exceeded by the induced mutant frequency at any concentration. No dose-response relationship was observed. Additionally, in experiment I and II colony sizing showed no clastogenic effects induced by the test item under the experimental condition (with and without metabolic activation).

Ethylmethanesulfonate (EMS), methylmethanesulfonate (MMS) and benzo[a]pyrene (B[a]P) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a)P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

In conclusion, in the described mutagencity test under the experimental conditions reported, the test item Reaction mass of 2-butylheptanoic acid and 2-ethylnonanoic acid and 2-methyldecanoic acid and 2-propyloctanoic acid is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A bacterial reverse mutation test using Salmonella typhimurium his- and Escherichia coli WP2 (uvrA-)(pKM101) was performed with the target substance 2 -hexyldecanoic acid according to OECD Guideline n°471 in compliance with Good Laboratory Practices (GLP).

Solutions obtained from the test item have been tested for their capacity to induce reverse mutation in four Salmonella typhimurium strains and one Escherichia coli WP2 (uvrA-)(pKM101) strain. This study was performed in the absence and presence of metabolic activation. Two independent assays were carried out. For assay n°1, various concentrations were put in contact with the strains in the absence and presence of a metabolic activation system (S9 -mix 10% (v/v)). For assay n°2, various concentrations were put in contact with test strains in absence and presence of a metabolic activation system (S9 -mix 10%(v/v)). For the two assays, negative and postive controls were carried out in parallel. Positive controls induced a significant increase in the number of revertant colonies compared to negative controls. There was no siginificant difference between the number of spontaneous reversions, the number of reversions obtained in the prositive controls (without and with metabolic activation), and the mean of corresponding experimental "historical" values obtained in the laboratory. These results validated the two tests. There was no evidence of any increase in the number of revertant colonies in the presence of the various concentrations of the test item (50, 150, 500, 1500, and 5000 µg/plate), without and with metabolic activation in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100, and in Escherichia coli WP2 (uvrA-)(pKM101). The test item was judged to be non-mutagenic in this test system under the described conditions.

In a supporting study with the source substance 2-butyloctanoic acid, the source substance was examined for mutagenic activity by assaying for reverse mutation in Salmonella typhimurium, strains TA1535, TA1537, TA98, TA100 and TA102. Experiments were performed both in the absence and presence of metabolic activation. The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose-level, in any tester strain, in the absence or presence of S9 metabolism. It was concluded that 2-butyloctanic acid did not induce reverse mutation in Salmonella typhimurium under the reported experimental conditions.

In another supporting study the source substance Reaction mass of n-undecanoic acid and 2-methyl-decanoic-acid and 2-ethyl-nonanoic-acid and 2-propyl-octanoic-acid and 2 -butyl-heptanoic-acid, was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and tester strain E. coli WP2 uvrA. The study was designed to be compliant with OECD Guideline No. 471 and in accordance with the OECD Principles of Good Laboratory Practices.

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. Toxic effects of the test item were noted in all tester strains used in experiment I and II. In experiment I toxic effects of the test item were observed at concentrations of 1.0 µL/plate and higher with and without metabolic activation depending on the particular tester strain. In experiment II toxic effects of the test item were noted at concentrations of 0.316 µL/plate and higher with and without metabolic activation, depending on the particular tester strain.

No biologically relevant increases in revertant colony number of any of the five tester strains were observed following treatment with the test substance.

Therefore, the test substance was considered to be non-mutagenic in this bacterial reverse mutation assay.

On the target substance no Chromosome aberration test is available.

The in vitro chromosome aberration study with the source substance Reaction mass of 2 -butylheptanoic acid and 2 -ethylnonanoic acid and 2 -methyldecanoic acid and 2 -propyloctanoic acid was conducted according to OECD Guideline 473 and in compliance with GLP.

The metaphases were prepared 21 h after start of treatment with the test item. The treatment interval was 4 h without and with metabolic activation in experiment I. In experiment II, the treatment interval was 21 h without and 4 h with metabolic activation. Duplicate cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations.

The following concentrations were evaluated for the microscopic analysis of chromosomal aberrations:

Experiment I without and with metabolic activation. 1.0, 2.0 and 3.0 mM

Experiment II without metabolic activation: 0.1, 0.25 and 0.5 mM and with metabolic activation: 1.0, 2.5, 3.0 and 3.5 mM.

No precipitation of the test item was noted without and with metabolic activation in all dose groups evaluated in both experiments.

In experiment I without and with metabolic activation cytotoxic effects of the test item were noted at 3 mM and higher considering the relative cell count. The relative cell count was decreased to 51% without metabolic activation, respectively to 65% with metabolic activation. In experiment II without metabolic activation, cytotoxic effects of the test item were observed at a test item concentration of 0.5 mM considering the relative mitotic index. With metabolic activation cytotoxic effects were observed at the concentrations 3.0 and 3.5 mM regarding the mitotic index.

In experiment I without and with metabolic activation and in experiment II without metabolic activation no biologically relevant increase of aberrant cells was noted at all concentrations evaluated. Moreover the aberration rates of all dose groups treated with the test item were within the historical control data of the negative control. In experiment II with metabolic activation no increase in chromosome aberrations was noted up to a concentration of 3.0 mM. The aberration rates of the concentrations up to 3.0 mM were within the historical control data of the negative control. At the highest concentration tested (3.5 mM), increased numbers of aberrations were noted with 10.5%. However, as this increase might be induced by cytotoxicity, this effect is considered as not biologically relevant and will not be considered for evaluation of results.

In experiments I and II without and with metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the negative controls.

The distinct increase in the number of structural chromosome aberrations induced by the positive controls (ethylmethanesulfonate, methylmethanesulfonate and cyclophosphamide) clearly demonstrates the sensitivity of the test system.

In conclusion, it can be stated that under the experimental conditions of the study, the test item did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line. Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test.

On the target substance no Mouse Lymphoma Mutation study is available.

The source substance Reaction mass of 2 -butylheptanoic acid and 2 -ethylnonanoic acid and 2 -methyldecanoic acid and 2 -proyloctanoic acid was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The selection of the concentrations used in the main experiments was based on data from the pre-experiment. In experiment I 1.3 mM (without metabolic activation) and 1.4 mM (with metybolic activation) were selected as the highest concentrations. In experiment II 0.5 mM (without metabolic activation) and 1.4 mM (with metabolic activation ) were selected as the highest concentrations. Experiment I without and with metabolic activation and experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as a 24 h long-term exposure assay.

The test item was investigated at the following concentrations:

Experiment I without metabolic activation: 0.05, 0.1, 0.2, 0.5, 0.7, 0.9, 1.1 and 1.3 mM and with metabolic activation: 0.02, 0.05, 0.1 ,0.2, 0.5, 1.0, 1.2 and 1.4 mM

Experiment II without metabolic activation: 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2 and 0.5 mM and with metabolic activation: 0.15, 0.3, 0.7, 0.9, 1.1, 1.2, 1.3 and 1.4 mM.

No precipitation of the test item was noted in the experiments. Growth inhibition was observed in experiment I and II without and with metabolic activation.

In experiment I without metabolic activation the relative total growth (RTG) was 21.8% for the highest concentration (1.3 mM) evaluated. The highest concentration evaluated with metabolic activation was 1.4 mM with a RTG of 10.1%. In experiment II without metabolic activation the relative total growth was 20.3% for the highest concentration (0.5 mM) evaluated. The two highest concentrations evaluated with metabolic activation were 1.3 and 1.4 mM with a RTG of 31.8% and 8.7% respectively.

In experiment I and II no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). The Global Evaluation Factor (GEF, defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories) was not exceeded by the induced mutant frequency at any concentration. No dose-response relationship was observed. Aditionally, in experiment I and II colony sizing showed no clastogenic effects induced by the test item under the experimental condition (with and without metabolic activation).

Ethylmethanesulfonate (EMS), methylmethanesulfonate (MMS) and benzo[a]pyrene (B[a]P) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a)P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

In conclusion, in the described mutagencity test under the experimental conditions reported, the test item Reaction mass of 2-butylheptanoic acid and 2-ethylnonanoic acid and 2-methyldecanoic acid and 2-propyloctanoic acid is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Justification for classification or non-classification

According to the classification criteria of regulation (EC) 1272/2008 2 -hexyldecanoic acid has not to be classified as mutagenic..