Isohexadecyl 12-[(1-oxooctadecyl)oxy]octadecanoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative with and without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 100, TA 98, TA 1538 and E. coli WP2 uvr A
Gene mutation in mammalian cells (OECD 476): negative in Chinse hamster lung fibroblasts (V79) and mouse lymphoma L5178Y cells with and without metabolic activation

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

09 Apr - 27 Jul 2010

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine Kinase locus (TK gene)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes were prepared from adult male Wistar rats which were treated with phenobarbital (89 mg/kg bw) and β-naphthoflavone (100 mg/kg bw).

Test concentrations with justification for top dose:

0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation; used at a concentration of 7.5 µg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation; used at concentration of 15 and 5 µg/mL for a 3 and 24 hours treatment period

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
For 3-hour exposure: Cells were exposed to the test substance in basic medium supplemented with 5% (v/v) heat-inactivated horse serum.
For 24-hour exposure: Cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heat-inactivated horse serum.
Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 µg/mL trifluorothymidine (TFT). Non selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) .

DURATION
- Exposure duration: In experiment 1 - 3 hours. Experiment 2 - 24 hours (- S9 mix) and 3 hours (+S9)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11-12 days

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT)
STAIN (for cytogenetic assays): 0.5 mg/mL of 3-[4,5-dimethylthiazol-2-yl]-2-yl]-2,5- diphenyltetrazolium bromide (MTT).

NUMBER OF REPLICATIONS: 1

NUMBER OF CELLS EVALUATED: Whole wells counted

DETERMINATION OF CYTOTOXICITY
- Method: Relative total growth

Evaluation criteria:

In addition to the criteria stated below, any increase of the mutation frequency will be evaluated for its biological relevance including a comparison of the results with the historical control data range.
The global evalulation factor (GEF) has been defined by the IWTG as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test substance is considered postive (mutagenic) in the mutation assay if it induces a mutation factor (MF) of more than MF(control) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range. The test substance will be considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
Thes test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The resullts are confirmed in an indepedently repeated test.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: 2-Ethylhexyl oleate was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test substance concentration for the dose range finding test was 333 µg/mL.
- Precipitation: 2-Ethylhexyl oleate precipitated in the exposure medium at concentrations of 100 µg/mL and above.

RANGE-FINDING/SCREENING STUDIES: In the dose finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 3 to 333 µg/mL in the absence of S9-mix with a 3 and 24-hour treatment period and in the presence of S9-mix with a 3-hour treatment period.
After 3 hours treatment and 24 and 48 hours subculture, no toxicity in the suspension growth was observed up to and including the highest test substance concentration of 333 µg/mL compared to the suspension growth of the solvent control both in the absence of S9-mix and presence of S9-mix.
After 24 hours of treatment and 24-hour subculture, in the absence of S9-mix, no toxicity in the relative suspension growth was observed up to test substance concentration of 333 µg/mL compared to the solvent control.


COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the solvent-treated control cultures were between the
minimum and maximum value of the historical control data range. See Table 1 to 4.

Remarks on result:

other: strain/cell type: mouse lymphoma L5178Y cells

Table 1. Experiment 1: Cytotoxic and mutagenic response of 2-Ethylhexyl oleate in the mouse lymphoma L5178Y test system.

Dose (µg/mL)	RSG (%)	CE^day2(%)	RS^day2 (%)	RTG (%)	Mutation Frequency x 10-6
					Total	Small	Large
	Without metabolic activation (3-hour treatment)
SC1	100	89	100	100	100	69	28
SC2	100	108	100	100	99	67	28
0.03	106	105	107	113	87	63	20
0.1	102	101	102	104	76	50	23
0.3	88	86	88	77	109	71	34
1	107	99	101	108	99	73	22
3	106	97	98	104	93	64	25
10	103	105	107	110	88	62	22
33	82	111	113	92	133	86	38
100^(1)	82	120	121	100	93	67	22
MMS	66	56	57	37	1463	939	292
	With 8% (v/v) metabolic activation (3-hour treatment)
SC1	100	65	100	100	68	41	26
SC2	100	67	100	100	58	32	25
0.03	96	66	100	96	73	45	26
0.1	102	63	95	97	71	39	30
0.3	93	67	102	94	71	46	24
1	107	66	100	107	71	40	29
3	108	58	88	95	74	53	20
10	107	53	80	85	74	50	22
33	95	62	94	89	74	43	29
100^(1)	100	54	81	81	68	44	23
CP	57	44	66	38	752	574	137

Note: all calculations were made without rounding off.

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent control = Ethanol; MMS Methylmethanesulfonate; CP = Cyclophosphamide.

(1) = 2-Ethylhexyl oleate precipitated in the exposure medium.

Table 2. Experiment 2: Cytotoxic and mutagenic response of 2-Ethyl oleate in the mouse lymphoma L5178Y test system.

Dose (µg/mL)	RSG (%)	CE^day2(%)	RS^day2 (%)	RTG (%)	Mutation Frequency x 10-6
					Total	Small	Large
	Without metabolic activation (24-hour treatment)
SC1	100	85	100	100	59	35	22
SC2	100	93	100	100	63	35	26
0.03	119	93	104	124	67	36	29
0.1	134	91	103	138	56	34	21
0.3	121	115	129	156	40	20	20
1	124	97	109	135	47	35	12
3	105	89	100	105	57	38	18
10	124	94	106	131	52	27	24
33	119	99	112	133	58	31	25
100^(1)	129	97	109	140	44	28	15
MMS	106	81	92	97	503	305	152
	With 12% (v/v) metabolic activation (3-hour treatment)
SC1	100	76	100	100	102	51	47
SC2	100	101	100	100	76	38	35
0.03	96	81	92	88	82	44	36
0.1	100	80	91	91	82	44	35
0.3	103	95	108	111	97	52	41
1	106	101	114	121	70	41	27
3	102	83	94	95	85	40	42
10	96	88	99	95	76	46	28
33	99	81	92	91	89	55	31
100^(1)	98	86	98	96	73	37	34
MMS	62	66	75	46	1337	776	310

Table 3. Historical control data of the spontaneous mutation frequencies of the solvent controls of the mouse lymphoma assay.

	-S9-mix		+S9-mix
	3-hour treatment	24-hour treatment
Range	[51-120] x 10^-6	[50-127] x10-^6	[50-170] x 10^-6
Mean	77 x 10^-6	80 x 10^-6	92 x 10^-6
SD	18 x 10^-6	19 x10^-6	33 x10^-6
n	88	82	141

SD = Standard deviation

n = Number of observation

The above mentioned historical control data range of the solvent controls were obtained by collecting all data of the solvent control values (media, DMSO, and ethanol) over the period of June 2006 to June 2009.

Table 4. Historical control data of the spontaneous mutation frequencies of the positive controls for the mouse lymphoma assay.

	-S9-mix		+S9-mix
	3-hour treatment	24-hour treatment
Range	[518-2052] x 10^-6	[578-1533] x10-^6	[724-3715] x 10^-6
Mean	1004 x 10^-6	1063 x 10^-6	1597 x 10^-6
SD	356 x 10^-6	232 x10^-6	712 x10^-6
n	45	34	81

 

The above mentioned historical control data range of the positive controls were obtained by collecting all data over the period of June 2006 to June 2009.

Conclusions:

CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

08 Mar - 15 Apr 1994

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 April 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Energie und Bundesangelegenheiten, Wiesbaden, Germany

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: minimal essential medium (MEM) supplemented with 10 % fetal calf serum (FCS)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes, by treatment with HAT-medium
- Doubling time 12 - 16 h in stock cultures

Metabolic activation:

with and without

Metabolic activation system:

Co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with 500 mg/kg bw Aroclor 1254 i.p. 5 days prior to sacrifice.

Test concentrations with justification for top dose:

10, 30, 60 and 100 µg/mL, with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol. The final concentration of ethanol in the culture medium did not exceed 1 % v/v.
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its non-toxicity to the cells.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethylmethanesulphonate (600 µg/mL in MEM without FCS, -S9) and 7,12-dimethylbenz(a)anthracene (3.85 µg/mL in DMSO, +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent):
Cloning efficiency: 7 days
Mutant selection: 9 and 12 days (experiments 2 and 1, respectively)
- Fixation time (start of exposure up to fixation or harvest of cells):
Cloning efficiency: 7 and 14 days
Mutant selection: 16 and 19 days (experiments 2 and 1, respectively)

Counting of colonies: the colonies were stained with 10 % methylene blue in 0.01 % KOH solution. Stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

SELECTION AGENT (mutation assays): thioguanine, 11 µg/mL medium (Sigma, Deisenhofen, Germany)

NUMBER OF REPLICATIONS: 1 replication in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: determination of concentration-related cloning efficiency on day 7 after start of exposure; determination of cell survival on day 14 after start of exposure (end of selection time)

Evaluation criteria:

The test substance is considered to be positive if it induces either a concentration-related increase of the mutant frequency or a reproducible positive response for one of the test points.
A test material producing neither a concentration-related increase in the mutant frequency nor a reproducible positive response in any of the test points is considered non-mutagenic in this system.
A significant response is:
1) test substance induces reproducibly with one of the concentrations a mutation frequency that is 3 times higher than the spontaneous mutation frequency in the experiment
2) test substance induces reproducible concentration-related increase of the mutation frequency. May also be considered in case a 3-fold increase of the mutant frequency is not observed.

In a case-by-case evaluation the decision depends on the level of the corresponding negative control data.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitation at limit of water solubility at 100 µg/mL observed.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Due to limited solubility of the test substance higher concentrations than 100 µg/mL for the cytogenetic evaluation were not feasible.
- Precipitation: observed at concentrations higher than 100 µg/mL

RANGE-FINDING/SCREENING STUDIES
Data on the pre-test were obtained from the chromosomal aberration study (IUCLID section 7.6.1: key, Emery, 1994, ChrAb, RL1)
A pre-test was performed to determine the toxicity of the test article. The test substance was dissolved in ethanol due to its solubility properties. The highest attainable concentration was 100 µg/mL, at which a slight precipitation was observed. No toxic effects were observed up to and including the highest dose level.

COMPARISON WITH HISTORICAL CONTROL DATA: yes (data not presented)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In parallel to the mutagenicity testing the cloning efficiency was tested. Without metabolic activation the relative cloning efficiencies were between 64.2 - 91.8 % in treated cells compared to control cells. With metabolic activation the relative cloning efficiencies were between 92.4 - 109.7 % in treated cells compared to control cells.

No statistically significant increase in gene mutations was observed (see Table 1 and 2). The positive controls were shown to be valid.

Table 1: mutagenicity data, experiment 1

	Concentration (µg/mL)	Metabolic activation	No. of mutant colonies after plating in TG medium* (mean of 5 flasks)	% cell survival	No. of mutant colonies per 10^6surviving cells	% cloning efficiency
Negative control	-	-	2.2	54	9.4	100.0
Vehicle control	-	-	1.0	66	3.9	100.0
EMS	600	-	89.6	50	416.4	73.8
Test substance	10	-	1.0	72	3.3	91.8
Test substance	30	-	2.0	72	6.8	72.0
Test substance	60	-	0.2	81	0.6	68.1
Test substance	100	-	2.8	65	10.2	64.2
Negative control	-	+	2.0	69	7.3	100.0
Vehicle control	-	+	3.2	68	11.4	100.0
DMBA	3.85	+	112.0	57	496.2	53.1
Test substance	10	+	5.4	78	16.1	92.4
Test substance	30	+	0.2	65	0.8	95.6
Test substance	60	+	1.4	67	5.4	97.3
Test substance	100	+	0.2	74	0.7	109.7

*only colonies with more than 50 cells 8 days after seeding in TG selection medium were scored

** ration of mean number of cells found after 7 days in normal growth medium / cell number seeded per flask

TG = Thioguanine

EMS = Ethylmethanesulphonate

DMBA = 7,12-dimethylbenz(a)anthracene

 

Table 2: mutagenicity data, experiment 2

	Concentration (µg/mL)	Metabolic activation	No. of mutant colonies after plating in TG medium* (mean of 5 flasks)	% cell survival	No. of mutant colonies per 10^6surviving cells	% cloning efficiency
Negative control	-	-	2.0	69	6.6	100.0
Vehicle control	-	-	1.8	64	6.4	100.0
EMS	600	-	145.8	60	496.9	61.5
Test substance	10	-	4.2	62	18.4	93.6
Test substance	30	-	1.0	63	4.1	107.8
Test substance	60	-	3.2	60	13.5	56.4
Test substance	100	-	0.8	59	3.5	61.4
Negative control	-	+	2.4	60	10.3	100.0
Vehicle control	-	+	2.2	61	9.0	100.0
DMBA	3.85	+	141.6	64	550.4	50.3
Test substance	10	+	1.4	61	5.7	104.8
Test substance	30	+	2.2	72	7.5	106.1
Test substance	60	+	0.6	69	2.1	102.8
Test substance	100	+	1.4	79	4.4	107.7

*only colonies with more than 50 cells 8 days after seeding in TG selection medium were scored

** ratio of mean number of cells found after 7 days in normal growth medium / cell number seeded per flask

TG = Thioguanine

EMS = Ethylmethanesulphonate

DMBA = 7,12-dimethylbenz(a)anthracene

Conclusions:

CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The Salmonella typhimurium strain TA 102 or an E. coli strain was not included, only 2-aminoacridine was used as the positive control with metabolic activation and the S-9 mix was not characterised with a mutagen requiring metabolic activation, the test substance purity was not specified, only µL/plate given.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1997

Deviations:

yes

Remarks:

S. typhimurium strain TA 102 or an E. coli strain not included, only 2-aminoacridine used as the positive control with metabolic activation and the S-9 mix was not characterised with a mutagen requiring metabolic activation

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 1538

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Experiment I and II: 1.0, 5.0, 10, 50 and 100 µL/plate (all strains), with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: the test substance was miscible to 1mL/mL in acetone and corn oil. The stock solution of the test substance was prepared in acetone, therefore acetone was selected as the vehicle

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (1 µg/plate, -S9, TA 1535, TA 100); 9-aminoacridine (50 μg/plate, -S9, TA 1537); 2-nitrofluorene (5 μg/plate, -S9, TA 98, TA 1538); 2-aminoanthracene (1.25 μg/plate, +S9,TA 1535, TA 1537, TA 98, TA 100, TA 1538)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: triplicates in each of two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (thinning of bacterial background lawn); reduction in the number of revertant colonies per plate

OTHER:
In a preliminary range-finding test, the cytogenicity of the test substance was assessed. TA 100 was treated with 0.05, 0.1, 0.5, 1.0, 5.0, 10, 50 and 100 µL/plate, with and without metabolic activation. The genotypes of the tester strains were confirmed by testing for histidine requirement and rfa-mutation, and TA and TA 100 were confirmed positive for the pKM101 plasmid. The viabilty of the tester strain was confirmed. The sterility of the S9-mix was checked before the beginning and the end of each experiment and was found to be satisfactory.

Evaluation criteria:

Evaluation criteria:
A response is considered positive if at least one strain has a dose that produces a mean reversion frequency that is 2 times or more greater than the mean reversion frequency of the corresponding solvent control plates and the response is dose dependent. The degree of toxicity will be considered in relation to the results. A response is considered equivocal if it does not fulfill the criteria of either negative or positive response and/or the study director does not consider the reponse to be either positive or negative.

Statistics:

Mean values and standard deviations were calculated for the number of revertants per plate.

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: the test substance was soluble up to 1 mL/mL in acetone and corn oil
- Precipitation: slight precipitation was observed in the range-finding assay from 50 µL/plate (TA 100) with and without metabolic activation and in the main assays with and withour metabiolic activation.

RANGE-FINDING/SCREENING STUDIES: cell viability was comparable between control and treated strains up to the highest concentration level of 100 µL/plate. Slight precipitation was observed in the range-finding assay from 50 µL/plate (TA 100) with and without metabolic activation.

Remarks on result:

other:

Table 1: Experiment 1

EXPERIMENT 1 (plate incorporation test)
S9-Mix	Without
Test item (µL/plate)	TA 98	TA 100	TA 1535	TA 1537	TA 1538
NC	26 ± 5.9	80 ± 4.0	21 ± 4.0	12 ± 3.6	10 ± .6
1.0	24 ± 2.3	80 ± 3.8	21 ± 1.5	13 ± 3.1	10 ± 3.8
5.0	24 ± 1.5	78 ± 1.5	19 ± 3.8	11 ± 2.9	9 ± 4.0
10	29 ± 5.9	69 ± 2.0	16 ± 3.5	11 ± 3.1	10 ± 2.6
50	27 ± 3.6*	85 ± 4.9*	16 ± 0.6*	9 ± 2.6*	10 ± 2.1*
100	23 ± 3.5*	82 ± 12.8*	20 ± 0.6*	9 ± 3.0*	9 ± 4.0*
2-NF	775 ± 71.5	-	-	-	857 ± 60.2
SA	-	476 ± 4.7	456 ± 16.7	-	-
9-AA	-	-	-	86 ± 10.8	-
S9-Mix	With
Test item (µL/plate)	TA 98	TA 100	TA 1535	TA 1537	TA 1538
NC	26 ± 6.0	57 ± 4.0	11 ± 1.7	13 ± 1.2	14 ± 1.7
1.0	31 ± 6.7	61 ± 0.6	13 ± 1.5	13 ± 2.3	14 ± 1.5
5.0	34 ± 1.2	75 ± 6.0	14 ± 4.0	11 ± 3.1	16 ± 7.2
10	32 ± 2.5	63 ± 4.4	16 ± 1.2	11 ± 2.6	11 ± 5.0
50	29 ± 4.0*	67 ± 2.0*	15 ± 0.6*	10 ± 3.6*	11 ± 4.9*
100	27 ± 3.6*	61 ± 1.7*	15 ± 1.5*	11 ± 2.1*	12 ± 0.6*
2AA	665 ± 43.9	716 ± 68.0	366 ± 1.5	138 ± 1.0	720 ± 114.1
*slight precipitate NC = Vehicle Control, acetone 2-NF: 2-nitrofluorene SA: sodium azide 9AA:9-aminoacridine 2AA: 2-aminoanthracene

 

Table 2: Experiment 2

EXPERIMENT 2 (plate incorporation test)
S9-Mix	Without
Test item (µL/plate)	TA 98	TA 100	TA 1535	TA 1537	TA 1538
NC	27 ± 2.6	66 ± 2.1	20 ± 0.6	11 ± 3.1	11 ± 1.2
1.0	24 ± 1.5	70 ± 2.6	19 ± 2.9	12 ± 2.9	11 ± 2.1
5.0	25 ± 3.1	71 ± 2.5	19 ± 2.1	13 ± 5.5	12 ± 2.0
10	28 ± 3.0	56 ± 2.1	19 ± 4.6	12 ± 2.5	12 ± 2.5
50	26 ± 4.7*	63 ± 5.5*	23 ± 1.2*	11 ± 1.2*	11 ± 1.7*
100	21 ± 3.2*	61 ± 3.8*	21 ± 2.6*	10 ± 2.5*	9 ± 0.6*
2-NF	781 ± 73.2	-	-	-	919 ± 97.6
SA	-	486 ± 31.3	462 ± 14.6	-	-
9-AA	-	-	-	100 ± 6.0	-
S9-Mix	With
Test item (µL/plate)	TA 98	TA 100	TA 1535	TA 1537	TA 1538
NC	33 ± 6.5	57 ± 6.6	17 ± 1.5	14 ± 1.5	13 ± 1.0
1.0	36 ± 4.0	57 ± 3.1	16 ± 5.5	12 ± 2.5	12 ± 0.6
5.0	36 ± 2.6	57 ± 8.9	14 ± 4.0	12 ± 2.0	12 ± 2.3
10	33 ± 3.1	58 ± 11.7	14 ± 1.2	12 ± 0.6	12 ± 3.0
50	37 ± 3.8*	58 ± 10.1*	15 ± 1.0*	10 ± 2.5*	15 ± 4.9*
100	33 ± 7.0*	60 ± 9.0*	12 ± 2.1*	13 ± 3.2*	13 ± 0.6*
2AA	699 ± 50.8	802 ± 97.2	344 ± 5.9	122 ± 8.1	673 ± 68.5
*slight precipitate NC = Vehicle Control, acetone 2-NF: 2-nitrofluorene SA: sodium azide 9AA:9-aminoacridine 2AA: 2-aminoanthracene

 

 

 

Conclusions:

CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

11 - 20 Mar 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9-mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

10, 33, 100, 333 and 1000 µg/plate, with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide, 9-aminoacridine, daunomycine, methylmethanesulfonate, 4-nitroquinoline-N-oxide, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (reduction of bacterial background lawn, increase in size of the microcolonies, reduction of the revertant colonies)

OTHER:
the results of the dose range-finding test were included as part of experiment 1

Evaluation criteria:

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following
criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory background historical range for each tester strain (see Table 1)
b) The positive control chemicals should produce responses in all tester strains which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least two times the concurrent vehicle control group mean
c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants compared to the solvent control value in any of the tester strains, either with or without metabolic activation. However, any plate with a mean plate count of less than 20 colonies is considered to be not significant and the result will be disregarded.
b) The positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation was observed in all strains, with and without metabolic activation, from 1000 µg/plate and above.

RANGE-FINDING/SCREENING STUDIES:
A dose range-finding test with strain TA100 and the WP2uvrA strain (both with and without S9- mix) was performed to select suitable doses for the main experiments. Eight concentrations (3, 10, 33, 100, 333, 1000, 3330, 5000 µg/plate) were tested in triplicate. The results were reported as a part of the first experiment of the mutation assay. The highest concentration of the test substance used in the main experiments was the level at which the test substance exhibited limited solubility. Precipitation was observed on the plates from 1000 µg/plate and above in both strains.

COMPARISON WITH HISTORICAL CONTROL DATA: yes, the spontaneous mutation rate of each tester strain per plate were within the characteristic spontaneous mutation range (see Table 1 under 'Any other information on materials and methods incl. tables'), with the exception of TA 100 without metabolic activation. The value was slightly outside the limit of the range, therefore the validity of the test was considered not to be affected.

ADDITIONAL INFORMATION ON CYTOTOXICITY: no cytotoxicity was observed at any concentration level, with or without metabolic activation (see Table 2 and 3).

Remarks on result:

other: all strains/cell types tested

Table 2: Results of experiment 1

 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± SD)
		Base-pair substitution type		Frameshift type
		TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537
-S9	ethanol	55 ± 3	12 ± 3	14 ± 6	16 ± 2	6 ± 1
-S9	3	55 ± 6	-	11 ± 2	-	-
-S9	10	63 ± 7	10 ± 4	10 ± 3	15 ± 6	7 ± 2
-S9	33	49 ± 7	8 ± 3	11 ± 1	16 ± 1	7 ± 4
-S9	100	59 ± 2	8 ± 1	12 ± 2	15 ± 3	5 ± 3
-S9	333	57 ± 7	7 ± 2	11 ± 6	17 ± 4	5 ± 3
-S9	1000 SP	70 ± 6	10 ± 3	10 ± 2	16 ± 1	4 ± 1
-S9	3300 SP	77 ± 9	-	12 ± 2	-	-
-S9	5000 SP	59 ± 13	-	10 ± 4	-	-
Positive controls, –S9	Name	MMS	SA	4-NQO	DM	9-AC
	Concentrations (μg/plate)	650	1	10	4	60
		529 ± 23	121 ± 18	291 ± 26	375 ± 36	185 ± 66
+S9	ethanol	71 ± 3	8 ± 2	10 ± 1	26 ± 2	8 ± 1
+S9	3	66 ± 10		11 ± 4
+S9	10	62 ± 3	9 ± 2	9 ± 4	25 ± 4	8 ± 3
+S9	33	64 ± 3	9 ± 3	14 ± 3	22 ± 2	6 ± 2
+S9	100	50 ± 8	9 ± 3	15 ± 3	24 ± 5	6 ± 2
+S9	333	45 ± 5	13 ± 4	10 ± 3	18 ± 4	6 ± 4
+S9	1000 SP	54 ± 8	12 ± 4	12 ± 6	23 ± 5	8 ± 1
+S9	3330 SP	54 ± 9	-	12 ± 1	-	-
+S9	5000 SP	51 ± 9	-	12 ± 2	-	-
Positive controls, +S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	1	2.5	5	2.5	2.5
		1182 ± 90	240 ± 8	88 ± 10	1047 ± 57	713 ± 149

MMS = methylmethanesulfonate

SA = sodium azide

4-NQO = 4-nitroquinoline-N-oxide

9-AC = 9-aminoacridine

DM = daunomycine

2-AA = 2-aminoanthracene

SP = slight precipitate

 

Table 3: Results of experiment 2

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± SD)
		Base-pair substitution type		Frameshift type
		TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537
-S9	ethanol	88 ± 10	10 ± 2	10 ± 6	22 ± 7	5 ± 2
-S9	10	94 ± 13	9 ± 3	9 ± 2	17 ± 2	7 ± 3
-S9	33	93 ± 3	8 ± 3	12 ± 2	17 ± 3	5 ± 1
-S9	100	90 ± 7	10 ± 3	9 ± 2	14 ± 5	5 ± 3
-S9	333	90 ± 9	6 ± 3	11 ± 3	15 ± 6	5 ± 1
-S9	1000 SP	77 ± 5	6 ± 3	9 ± 4	14 ± 4	7 ± 4
Positive controls, –S9	Name	MMS	SA	4-NQO	DM	9-AC
	Concentrations (μg/plate)	650	1	10	4	60
		714 ± 49	152 ± 10	936 ± 118	598 ± 16	251 ± 100
+S9	ethanol	99 ± 8	8 ± 4	11 ± 7	15 ± 1	7 ± 3
+S9	10	98 ± 10	8 ± 3	7 ± 1	17 ± 3	4 ± 2
+S9	33	103 ± 21	6 ± 3	8 ± 2	27 ± 6	7 ± 1
+S9	100	98 ± 9	8 ± 3	13 ± 7	21 ± 7	6 ± 2
+S9	333	93 ± 6	7 ± 5	8 ± 3	20 ± 5	8 ± 2
+S9	1000 SP	112 ± 2	9 ± 3	8 ± 1	23 ± 1	4 ± 1
Positive controls, +S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	1	2.5	5	2.5	2.5
		2235 ± 60	322 ± 15	406 ± 20	1732 ± 115	703 ± 41

 

MMS = methylmethanesulfonate

SA = sodium azide

4-NQO = 4-nitroquinoline-N-oxide

9-AC = 9-aminoacridine

DM = daunomycine

2-AA = 2-aminoanthracene

SP = slight precipitate

 

Conclusions:

CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Micronucleus assay (OECD 474): negative in mouse bone marrow cells

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Jun - 2 Aug 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

1000 immature erythrocytes/animal were scored for micronucleated immature erythrocytes which is fewer than the 4000 required, the highest dose level was more than four times higher than the recommended limit dose, the purity of the test substance was not specified.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted Sep 2014

Deviations:

yes

Remarks:

fewer than 4000 immature erythrocytes/animal were scored for micronucleated immature erythrocytes

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, USA
- Age at study initiation: 49 days
- Weight at study initiation: 27 - 31 g (males), 20 - 26 g (females)
- Assigned to test groups randomly: yes, under following basis: according to weight
- Fasting period before study: no
- Housing: three (range-finding study) or five (main study) animals per cage, with hardwood chip bedding changed at least twice per week
- Diet: Purina certified rodent chow (Code No. 5002, lot No. Apr 13 94 1A74), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 24
- Humidity (%): 57 - 69
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: 0.5 mL of the test substance was miscible in 0.5 mL acetone and 0.5 mL corn oil. Corn oil was selected as the vehicle.
- Lot/batch no. (if required): D09B (CPC International Inc.)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was dissolved in the appropriate volume of corn oil just before dosing. The dosing volume was 10 mL/kg bw.

Duration of treatment / exposure:

not applicable

Frequency of treatment:

single treatment

Post exposure period:

24, 48 and 72 h

Remarks:

Doses / Concentrations:
2.0, 5.0 and 10 mL/kg bw
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
1720, 4300 and 8600 mg/kg bw
Basis:
other: calculated based on a density of 860 mg/mL

No. of animals per sex per dose:

5/treatment period

Control animals:

yes, concurrent vehicle

Positive control(s):

triethylenemelamine
- Justification for choice of positive control(s): triethylenemelamine causes micronuclei
- Route of administration: intraperitoneal injection
- Doses / concentrations: 1.0 mg/kg bw

Tissues and cell types examined:

Tissue: bone marrow
Cell type: bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A range-finding study was performed to find the maximum tolerated dose. Dose groups, each comprising 3 males and 3 females, received a single dose of the test substance. The animals were observed for 3 days, during which mortality and physical condition were recorded daily and body wegiht was recorded on Day 1 prior to administration and on the day of sacrifice.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The animals were dosed once and sacrificed by cervical dislocation at 24, 48 or 72 h (test substance and vehicle control groups) or 24 h only (positive control group).

DETAILS OF SLIDE PREPARATION:
The femur bone was removed, and the bone marrow canal exposed and flushed with a small volume of foetal calf serum. The cell suspension was collected and centrifuged at 800 rpm for 5 min. The supernatant was removed, leaving approximately 0.1 mL above the pellet. The cells in the sediment were resuspended by flicking the tube until a homogenous suspension was observed. A drop of the cell suspension was placed on the end of a precleaned slide and the drop was spread along the length of the slide. The preparations were then air-dried, fixed for 15 min in methanol and air-dried again. The slides were stained using Wright-Giemsa stain for 3 minutes, rinsed in distilled water and allowed to air-dry. The dry slides were mounted in permount with a coverslip, the backsides were cleaned with methanol.

METHOD OF ANALYSIS:
The number of micronucleated polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) was determined in 1000 erythrocytes.
The number of micronuleated PCE per 1000 PCE were determined (4000 should be counted according to the relevant guideline)

Evaluation criteria:

The criteria for a valid test were: (1) in the vehicle control, the average number of MPCE per 1000 PCE should not exceed 5; (2) in the positive control, the increase in the average number of MPCE per 1000 PCE over the average number of MPCE for the vehicle control should be statistically significant; (3) at least three animals from each sex must be alive at the time of sacrifice for each dose level.
The test substance was considered to have caused a positive response in this assay if: (1) the test article shows a positive dose-response trend and a statistically significant increase over that of the concurrent vehicle control in the number of MPCE at one or more dose levels. In the event that the test substance caused a statistically significant increase in the number of MPCE due to an unusually low number of MPCE (less than 0.05%) in the concurrent vehicle control, the data from that dose may be compared to historical vehicle control data; (2) in the event there is no positive dose-response trend, at least 2 consecutive test doses show a statistically significant increase in the number of MPCE.
The test substance was considered to have caused a negative response if no indication of a positive dose-response trend was observed and none of the test doses had a statistically significant increase in the number of MPCE when compared to the vehicle control.

Statistics:

Data were analysed separately for male and female animals. Unless otherwise indicated, the frequency of micronucleated PCE in each dose group was compared to that in the respective vehicle control using a 2-tailed Student’s t-test. Results were considered significant if the p-value is ≤ 0.05.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 0.01, 0.05, 0.1, 0.5, 1.0, 2.0 and 10 mL/kg bw
- Solubility: 0.5 mL of the test substance was only miscible in 0.5 mL of acetone and corn oil, showing a minimum miscible concentration of 1 mL/mL for each vehicle.
- Clinical signs of toxicity in test animals: one male administered 0.1 mL/kg bw died by accident on Day 1 due to being squeezed between the wires on top of the cage. No clinical signs were observed during the study period. the body weight increase was as expected for this species and strain under the current study conditions.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes from the bone marrow of test substance treated animals (see Table 1 and 2 under 'Any other results incl. tables). The positive control substance (cyclophosphamide) induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes, showing that the positive control was valid.
- Ratio of PCE/NCE (for Micronucleus assay): The animals of the groups that were treated with the test substance did not show a decrease in the ratio of polychromatic to normochromatic erythrocytes compared with the vehicle controls. The groups that were treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared with the vehicle controls.

Table 1: Results of the in vivo micronucleus assay in male animals

			Mean PCEs / 1000 NCEs at sampling time			Total micronuclei per 1000 PCEs at sampling time
Exposure group	Number of animals	Dose [mL/kg]	24 h	48 h	72 h	24 h	48 h	72 h
Vehicle control (corn oil)	5	10	1.38	1.36	1.71	0.6	0.4	0.4
Positive control (triethylenemelamine)	5	1.0 mg/kg bw	0.38	n.d.	n.d.	58.8*	n.d.	n.d.
Test substance	5	2.0	1.79	1.51	1.46	0.6	0.0	0.6
Test substance	5	5.0	1.30	1.56	1.88	0.4	0.4	0.4
Test substance	5	10	1.22	1.26	1.55	0.8	0.4	0.4

n.d. = not determined; *statistically significant (p<0.001);

 

 

Table 2: Results of the in vivo micronucleus assay in female animals

			Mean PCEs / 1000 NCEs at sampling time			Total micronuclei per 1000 PCEs at sampling time
Exposure group	Number of animals	Dose [mL/kg]	24 h	48 h	72 h	24 h	48 h	72 h
Vehicle control (corn oil)	5	10	1.37	1.84	1.71	0.6	0.8	0.4
Positive control (triethylenemelamine)	5	1.0 mg/kg bw	0.43	n.d.	n.d.	65.2*	n.d.	n.d.
Test substance	5	2.0	1.44	1.70	1.46	0.2	0.6	0.6
Test substance	5	5.0	1.03	1.38	1.88	0.4	1.0	0.4
Test substance	5	10	1.20	1.37	1.55	1.0	0.4	0.4

n.d. = not determined; * statistically significant (p<0.001);

Conclusions:

CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Justification for read-across

There are no data available on the genetic toxicity of Isohexadecyl 12 -[(1 -oxooctadecyl)oxy]octadecanoate (CAS 97338-28-8). The assessment was therefore based on studies conducted with analogue substances as part of a read-across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

Genetic toxicity (mutagenicity) in bacteria in vitro

CAS 90052-75-8

The in vitro genetic toxicity of 2-octyldodecyl 12-[(1-oxooctadecyl)oxy]octadecanoate was assessed in a bacterial reverse mutation assay (Ames test), performed similar to OECD guideline 471 and under GLP conditions (Ames, 1994). The plate incorporation method was applied, using S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100, at concentrations up to 100 µL/plate, with and without metabolic activation. No S. typhimurium strain TA 102 or E. coli strain was included in the study. No cytotoxicity was observed, but slight precipitation was noted in the range-finding assay and main test with TA 100 from 50 µL/plate with and without metabolic activation. The negative and positive controls were shown to be valid. The test substance did not induce an increase in reversions in the S. typhimurium strains tested, with or without metabolic activation.

CAS 93803-87-3

The in vitro genetic toxicity of 2-octyldodecyl isooctadecanoate (CAS 93803-87-3) was assessed in a bacterial reverse mutation assay (Ames test) (Ames, 1998), performed according to OECD 471. S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and E. coli WP2 uvr A were exposed to the test substance at concentrations up to 1000 µg/plate. Precipitation was observed in the medium from 1000 µg/plate and above in all strain, with and without metabolic activation. The negative and positive control was shown to be valid. The test substance did not induce reversions in the S. typhimurium strains or E. coli strain, with or without metabolic activation.

Genetic toxicity (mutagenicity) in mammalian cells in vitro

CAS 3687-45-4

An in vitro mammalian cell gene mutation assay was performed using Oleyl oleate, according to OECD guideline 476 and under GLP conditions (HPRT, 1994). Two separate experiments were performed. Chinese hamster lung fibroblast (V79) cells were treated with Oleyl oleate at concentrations of up to 100 µg/mL for 4 hours, with and without metabolic activation. After an expression time of 7 days in growth medium, cells were incubated for 9 or 12 days with 6 -thioguanine as selection agent for forward mutation at the HPRT locus. Precipitation was seen at concentrations of 100 µg/mL and higher, while no cytotoxicity was observed at any concentration level. The positive and negative controls were valid and within the range of historical control data. No significant increase in mutation frequency was observed, with and without metabolic activation.

CAS 26399-02-0

An in vitro mammalian cell gene mutation assay was performed with 2-ethylhexyl oleate (CAS 26399-02-0) according to OECD guideline 476 and under GLP conditions (MLA, 2010). Mouse lymphoma L5178Y cells were exposed to the test substance in ethanol at concentrations up to 100 μg/mL, in the absence and presence of metabolic activation. In experiment 1, the exposure time was 3 hours with and without metabolic activation, while in experiment 2 the exposure time was 3 hours with metabolic activation and 24 hours without metabolic activation. Precipitation was observed at concentrations of 100 µg/mL and above. The positive and negative controls were valid and within the range of historical control data. No significant increase in mutation frequency was observed.

Genetic toxicity in vivo

CAS 90052-75-8

An in vivo mammalian erythrocyte micronucleus test was performed according to OECD guideline 474 and under GLP conditions, using 2-octyldodecyl 12-[(1-oxooctadecyl)oxy]octadecanoate (key, 1998). 5 CD-1 mice/sex/dose were administered 2.0, 5.0 and 10 mL/kg bw of the test substance via gavage and sacrificed after 24, 48 or 72 h. A vehicle control group was sacrificed after 24, 48 or 72 h, while the positive control group was sacrificed after 24 h. Bone marrow cells from the femur were extracted, and slides were prepared and stained using Wright-Giemsa stain. 1000 immature erythrocytes/animal were scored for micronucleated immature erythrocytes which is fewer than the 4000 required. No increase in the frequency of micronucleated polychromatic erythrocytes was observed. The treatment groups did not show a decrease in the ratio of polychromatic to normochromatic erythrocytes, compared with the vehicle control groups. No toxicity was observed up to and including the limit dose of 10 mL/kg bw (equivalent to 8600 mg/kg bw/day based on a density of 0.86). The positive control substance triethylenmelamine) was shown to be valid. The test substance did not cause genetic toxicity under the conditions of this test.

Overall conclusion for genetic toxicity

There is no available data on the in vitro or in vivo genetic toxicity of the target substance Isohexadecyl 12-[(1-oxooctadecyl)oxy]octadecanoate. Therefore, analogue read-across from source substances was applied from in vitro studies on genetic toxicity in bacterial cells and in mammalian cells, and from an in vivo study, using 4 source substances. The results of the available studies were consistently negative. Based on the available data and following the analogue approach, Isohexadecyl 12-[(1-oxooctadecyl)oxy]octadecanoate (CAS 97338 -28 -8) is not expected to be mutagenic.

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied toIsohexadecyl 12-[(1-oxooctadecyl)oxy]octadecanoate(CAS 97338-28-8), data will be generated from data for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

Therefore, based on the available data on analogue read-across approach, the results on genetic toxicity for the source substances do not meet the classification criteria according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.