Dodecyl laurate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471, read across): negative with and without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 1538, TA 100 and TA 98 and TA 102.

In vitro chromosome aberration (OECD 473, read across): negative in primary human peripheral lymphocytes with and without metabolic activation.

In vitro gene mutation in mammalian cells (OECD 476, read across): negative in mouse lymphoma L5178Y cells with and without metabolic activation.

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:

11 Nov 1999 - 13 Mar 2000

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:

yes

Remarks:

only 2-AA used as positive control in the presence of S9-mix

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

certified by the Hessische Ministerium für Umwelt, Energie, Jugend, Familie und Gesundheit

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

Additional strain / cell type characteristics:

other: rfa-; uvrB- (R+ for TA 98 and TA 100)

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: rfa-; uvrB+; R+

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and β-naphthoflavone.

Test concentrations with justification for top dose:

Experiment 1 and 2: 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment 2: 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (MERCK, D-64293 Darmstadt; purity > 99%)
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 10 µg/plate sodium azide (TA 1535 and TA 100; -S9); 4-nitro-o-phenylene-diamine (10 µg/plate in TA 98 and 50 µg/plate in TA 1537; -S9); 5.0 µl/plate methylmethanesulfonate (TA 102; -S9); 2.5 µg/plate 2-aminoanthracene (10.0 µg/plate in TA 102) (+S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (first experiment); preincubation (second and third experiment)

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in three independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn

Evaluation criteria:

A test item is considered positve if either a dose related increase in the number of revertants or a biologically relevant increase for at least one test concentration is induced. A test item producing neither a dose related increase in the number of revertants nor a biologically relevant positive response at any one of the test points is considered non-mutagenic in the test system.
A biologically relevant response is described as follows:
A test item is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98, TA 100 and TA 102 or thrice in strains TA 1535 and TA 1537. Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test item regardless whether the highest dose fulfilled the criteria described above or not.

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100, TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA: yes

Table 1. Test results without S9-mix

Without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates; 2 or 3 independent experiments)
		Base-pair substitution type							Frameshift type
		TA 100		TA1535			TA102		TA98		TA1537
Experiment		I	II	I	II	III	I	II	I	II	I	II
	0	132	58	19	11	26	184	208	27	15	24	18
–	Solvent control	114	62	16	13	26	152	201	26	14	24	22
–	33	104	69	11	12	-	173	198	25	14	28	24
–	100	123	71	17	12	-	167	191	25	10	27	22
–	333	127	68	23	12	25	178	280	20	13	30	23
–	1000	117	58	14	26	25	169	183	23	13	31	21
–	2500	115	68	16	35	23	119	266	20	7	23	25
–	5000	122	63	16	32	25	176	270	22	11	23	20
Positive controls, –S9	Name	SA		SA			MMS		4NOPD		4NOPD
	Concentrations (μg/plate)	10.0		10.0			5 µL/plate		10.0		50.0
	Mean No. of colonies/plate (average of 3)	1490	1150	1075	1210	760	1387	1270	389	250	86	95

Table 2. Test results with S9-mix

With S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates; 2 independent experiments)
		Base-pair substitution type							Frameshift type
		TA 100		TA1535			TA102		TA98		TA1537
Experiment		I	II	I	II	III	I	II	I	II	I	II
+	0	143	96	8	11	-	192	227	45	26	26	20
+	Solvent control	129	70	10	10	-	154	215	36	19	31	22
+	33	118	72	9	8	-	144	201	46	20	28	24
+	100	118	76	8	9	-	166	219	46	24	34	24
+	333	131	80	6	10	-	170	220	41	17	32	26
+	1000	125	50	9	6	-	186	211	49	17	26	17
+	2500	122	75	5	8	-	154	172	55	17	23	28
+	5000	128	77	11	6	-	153	217	45	22	33	27
Positive controls, +S9	Name	2-AA		2-AA			2-AA		2-AA		2-AA
	Concentrations (μg/plate)	2.5		2.5			10.0		2.5		2.5
	Mean No. of colonies/plate (average of 3)	1803	536	231	151	-	726	1497	892	536	117	169

SA = Sodium azide

4NOPD = 4-nitro-o-phenylene-diamine

MMS = Methyl methane sulfonate

2-AA = 2-Aminoanthracene

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 bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

25 Jul - 02 Aug 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

incomplete strain selection

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Genes involved in Histidine synthesis

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium 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:

8, 40, 200, 1000 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tween 80/water (bidest.)
The suspension medium was choosen according to solubility properties tested in a preliminary experiment.

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

Tween 80

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

2 µg/plate for TA 100 and TA 1535 without metabolic activation

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

Tween 80

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

80 µg/plate for TA 1537 without metabolic activation

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

Tween 80

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine

Remarks:

40 µg/plate for TA 98 and TA 1538 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

2.5 µg/plate for TA 1535, TA 1537 and 5 µg/plate for TA 98, TA 100, TA 1538 (with S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn

OTHER: The spontaneous mutation rates of each tester strain were within the characteristic spontaneous mutation rates

Evaluation criteria:

The test material may be considered positive in this test system if the following criteria are met: For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.
All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.

Statistics:

Means and standard deviations were calculated

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

Table 1: Mutagenicity on bacteria - experiment I

 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		TA 1535	TA100	TA1537	TA1538	TA98
-	Buffer	8	102	10	11	30
-	Solvent (Tween80/H2O)	7	99	9	8	29
-	8	4	110	9	10	28
-	40	6	100	11	6	32
-	200	12	112	8	10	28
-	1000	12	97	10	11	31
-	5000	9	106	9	11	30
Positive controls - S9	Name	SA	SA	9AA	4ND	4ND
	Concentrations (μg/plate)	2	2	80	40	40
	Number of colonies/plate	297	311	413	1677	830
+	Buffer	6	93	8	14	26
+	Solvent (Tween80/H2O)	8	94	8	12	37
+	8	7	94	7	21	36
+	40	8	104	8	10	35
+	200	6	97	9	12	37
+	1000	8	97	7	9	40
+	5000	6	95	7	13	37
Positive controls + S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2.5	5	2.5	5	5
	Number of colonies/plate	107	1870	208	1566	1720

 

9AA = 9-Aminoacridine

2AA = 2-Aminoanthracene

SA = Sodium Acide

4ND = 4-Nitro-o-phenylendiamine

 

 Table 2: Mutagenicity on bacteria - experiment II

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		TA 1535	TA100	TA1537	TA1538	TA98
	Buffer	9	99	7	9	30
-	Solvent (Tween80/H2O)	11	100	9	10	32
-	8	8	101	8	11	33
-	40	6	105	7	10	37
-	200	9	112	8	11	37
-	1000	8	107	10	12	39
	5000	13	100	11	10	32
Positive controls - S9	Name	SA	SA	9AA	4ND	4ND
	Concentrations (μg/plate)	2	2	80	40	40
	Number of colonies/plate	270	270	296	1648	1359
+	Buffer	9	108	9	11	35
+	Solvent (Tween80/H2O)	11	104	11	14	30
+	8	7	105	9	13	32
+	40	8	105	9	11	32
	200	7	121	5	11	38
+	1000	8	120	8	14	32
+	5000	7	112	7	10	41
Positive controls + S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2.5	5	2.5	5	5
	Number of colonies/plate	137	1774	253	1419	1810

 

9AA = 9-Aminoacridine

2AA = 2-Aminoanthracene

SA = Sodium Acide

4ND = 4-Nitro-o-phenylendiamine

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

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

06 - 15 Sep 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

No TA 102 or E.coli strains were tested.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

no TA 102 or E.coli strains were tested

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

no TA 102 or E.coli strains were tested

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 500 mg/kg bw Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1: 8, 40, 200, 1000 and 5000 µg/plate without metabolic activation; 16, 80, 400, 1000 and 5000 µg/plate with metabolic activation
Experiment 2: 8, 40, 200, 1000 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tween 80/aqua bidest.
- Justification for choice of solvent/vehicle: the vehicle was chosen according to the solubility properties tested before the start of the study

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (2.5 µg/plate, ± S9, TA 100, TA 1535); 9 -aminoacridine (80 µg/plate, ± S9, TA 1537); 4 -nitro-o-phenylenediamine (40 µg/plate, ± S9, TA98, TA 1538); 2 -aminoanthracene (5.0 µg/plate ± S9, TA 1535, TA 1537; 2.5 µg/plate, ± S9, TA 98, TA 100)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 12 h
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER: The test batches containing S9-mix were controlled for sterility by adding 0.5 ml S9-mix to untreated agar plates. The microsomal enzyme activity was examined with 2-amino-anthracene and benzo(a)pyrene on TA 98 and in a cytogenetic test with cyclophosphamide.

Evaluation criteria:

A combination of the following criteria was considered as a positive result:
- The plate background of non-reverted bacteria did not show any growth reduction versus the respective negative controls
- The spontaneous mutation rates of each tester strain per plate were within the characteristic spontaneous mutation range (see Table 1 under 'any other information on materials and method including tables')
- As a rule, the positive control showed mutation rates exceding the control values of TA 100 at least by the factor 2.0 and those of the other tester strains at least by the factor 3.0
- At more than one dose tested, the test substance caused at least a 2.0 fold increase in comparison with the negative controls in the tester strain S. typhimurium TA 100. For the other tester strains used, an increase in the mutation rate of more than 3.0 above the corresponding negative controls was considered positive.

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:

cytotoxicity

Remarks:

at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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).

ADDITIONAL INFORMATION ON CYTOTOXICITY: cytotoxicity was observed at 5000 µg/plate

Table 2: Test 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	TA 1538	TA 98	TA 1537
Negative controls, –S9	Culture medium and Tween 80/aqua bidest.	64.4 ± 11.4	10.0 ± 3.5	9.6 ± 2.9	23.8 ± 3.1	5.5 ± 1.3
–	8	67.6 ± 4.5	13.3 ± 2.5	10.0 ± 1.0	29.0 ± 4.3	9.6 ± 2.8
-	40	78.3 ± 12.0	9.0 ± 4.0	10.0 ± 1.0	29.3 ± 1.5	5.0 ± 2.6
-	200	79.5 ± 23.3*	7.0 ± 3.0	9.6 ± 2.5	22.6 ± 4.7	8.0 ± 6.0
–	1000	80.6 ± 17.0	8.6 ± 3.5	11.6 ± 3.2	31.0 ± 2.6	6.0 ± 1.0
–	5000	55.6 ± 9.4	8.6 ± 2.3	8.3 ± 0.5	29.6 ± 5.5	8.6 ± 2.8
Positive controls, –S9	Name	SA	SA	4-NP	4-NP	9-AA
	Concentrations (μg/plate)	2	2	40	40	80
	Mean No. of colonies/plate (average of 3 ± SD)	266.3 ± 32.3	573.0 ± 32.6	1871.3 ± 40.8	968.6 ± 144.3	218 ± 100.9
Positive controls, –S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	5	2.5	5	5	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	56.5 ± 2.1*	10.6 ± 1.1	16.6 ± 4.0	26.3 ± 7.2	7.0 ± 3.4
Negative controls, +S9	Culture medium and Tween 80/aqua bidest.	84.6 ± 17.9	12.0 ± 2.5	11.3 ± 1.8	18.0 ± 3.5	7.5 ± 3.3
+	16	71.3 ± 2.0	14.0 ± 2.6	14.3 ± 0.5	22.3 ± 3.5	4.6 ± 2.0
+	80	81.3 ± 11.5	10.3 ± 1.5	9.3 ± 0.5	23.6 ± 2.0	7.0 ± 3.0
+	400	89.3 ± 2.0	13.3 ± 5.5	13.6 ± 4.9	24.0 ± 3.6	7.0 ± 1.7
+	1000	94.6 ± 12.5	13.0 ± 2.6	9.0 ± 2.0	22.6 ± 2.0	4.0 ± 2.6
+	5000	100.0 ± 3.6	15.6 ± 3.0	9.0 ± 1.0	15.0 ± 3.0	5.0 ± 4.0
Positive controls, +S9	Name	SA	SA	4-NP	4-NP	9AA
	Concentrations (μg/plate)	2	2	40	40	80
	Mean No. of colonies/plate (average of 3 ± SD)	140.0 ± 18.7	20.6 ± 2.5	19.6 ± 2.8	721.3 ± 38.8	196.0 ± 67.6
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	5	2.5	5	5	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	1100.3 ± 55.1	438.0 ± 33.4	162.0 ± 22.5	884.0 ± 114.0	46.3 ± 9.0

9-AA = 9-aminoacridine

4-NP = 4-nitro-o-phenylenediamine

SA = sodium azide

2AA = 2-Aminoanthracene

* contamination of 1 plate

Table 3: Test 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	TA 1538	TA 98	TA 1537
Negative controls, –S9	Culture medium and Tween 80/aqua bidest.	113.1± 12.7	9.8± 3.7	11.8± 3.3	47.6± 4.4	8.0± 1.0
–	8	108.6± 7.0	10.0± 2.8	9.6± 3.7	48.6± 12.5	7.6± 3.0
-	40	109.6± 6.4	9.3± 4.3	11.3± 0.5	43.3± 8.6	6.6± 1.1
-	200	99.5± 6.3	14.6± 5.6	13.0± 6.0	47.0± 11.5	7.0± 2.6
–	1000	115.0± 14.1	11.0± 1.1	12.0± 3.4	49.0± 7.2	9.0± 2.0
–	5000	114.3± 9.2	6.3± 2.8	9.6± 2.0	44.0± 7.2	11.0± 3.6
Positive controls, –S9	Name	SA	SA	4-NP	4-NP	9-AA
	Concentrations (μg/plate)	2	2	40	40	80
	Mean No. of colonies/plate (average of 3 ± SD)	645.3± 56.1	388.6± 15.9	2167.3± 101.2	1103.3± 162.4	91.0± 19.5
Positive controls, –S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	5	2.5	5	5	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	107.3± 6.6	7.6± 2.3	18.6± 8.5	54.6± 3.2	9.6± 2.5
Negative controls, +S9	Culture medium and Tween 80/aqua bidest.	109.0± 10.0	15.1± 4.0	24.1± 1.9	61.3± 7.9	6.8± 2.9
+	8	92.3± 17.0	11.0± 2.6	27.3± 2.5	69.6± 14.5	9.6± 0.5
+	40	116.0± 7.0	12.3± 3.2	29.6± 1.5	59.3± 3.2	7.0± 1.7
+	200	104.0± 16.3	12.6± 4.9	27.0± 6.5	68.6± 8.0	7.3± 1.1
+	1000	118.6± 5.1	14.6± 5.5	25.3± 3.5	70.6± 8.0	7.6± 3.0
+	5000	113.6± 25.7	9.3± 3.0	28.6± 1.5	75.3± 3.2	12.6± 1.5
Positive controls, +S9	Name	SA	SA	4-NP	4-NP	9AA
	Concentrations (μg/plate)	2	2	40	40	80
	Mean No. of colonies/plate (average of 3 ± SD)	202.0± 16.0	178.3± 7.6	1487.0± 128.0	736.3± 7.9	65.0± 15.0
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	5	2.5	5	5	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	1337.6± 47.5	194.6± 24.9	1515.0± 171.6	1100.6± 47.5	87.6± 17.6

9-AA = 9-aminoacridine

4-NP = 4-nitro-o-phenylenediamine

SA = sodium azide

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 cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 Apr - 10 May 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix

Test concentrations with justification for top dose:

3, 10 and 33 µg/mL
At a concentration of 33 µg/mL and above precipitation was seen in the culture medium.

Vehicle / solvent:

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

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with rat liver S9-mix; 10 µg/mL

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without rat liver S9-mix; 0.5 µg/mL for a 3 h exposure period, 0.2 µg/mL for a 24 h exposure period and 0.1 µg/mL for a 48 h exposure period

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3, 24 and 48h
- Fixation time (start of exposure up to fixation or harvest of cells): 3h treatment: 24 and 48h; 24h treatment: 24h; 48h treatment: 48h

SPINDLE INHIBITOR (cytogenetic assays): colchicine 0.5 µL/mL medium
STAIN (for cytogenetic assays): Giemsa 5% (v/v) in tap water

NUMBER OF REPLICATIONS: 2 (duplicates at the 3h-exposure time)

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if: a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations. b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations. The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

Chi-square test, one-sided, p < 0.05

Key result

Species / strain:

lymphocytes: cultured human peripheral lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At a concentration of 33 µg/mL 2-ethylhexyl oleate precipitated in the culture medium
- Other confounding effects: 2-ethylhexyl oleate showed no significant effects on the number of polyploid cells

RANGE-FINDING/SCREENING STUDIES: The highest concentration analysed was selected based on the solubility of the test substance in the culture medium (3 h and 24 h continuous exposure) or on toxicity, inhibition of the mitotic index of about 50% or greater (48 h continuous exposure).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No cytotoxicity was observed in the duplicate cultures of the 3h exposure time.

Table 1: Cytotoxic and Genotoxic observations

Test item	Concentration	Mitotic Index	Aberrant cells in %
	in µg/mL	in %	with gaps	without gaps
Exposure period 3 h, fixation time 24 h, without S9 mix
Ethanol	1.0% (v/v)	100	2	2
MMC	0.5	67	31	30
Test substance	3	99	1	1
	10	98	2	2
	33	92	1	1
Exposure period 3 h, fixation time 24 h, with S9 mix
Ethanol	1.0% (v/v)	100	1	1
CP	0.5	51	38	38
Test substance	3	101	1	1
	10	108	0	0
	33	103	3	3
Exposure period 24 h, fixation time 24 h, without S9 mix
Ethanol	1.0% (v/v)	100	1	1
MMC	0.2	54	34	33
Test substance	3	99	1	1
	10	103	3	3
	33	70	4	3
Exposure period 48 h, fixation time 48 h, without S9 mix
Ethanol	1.0% (v/v)	100	2	0
MMC	0.1	120	51	49
Test substance	3	108	1	0
	10	100	0	0
	33	99	2	2
Exposure period 3 h, fixation time 48 h, with S9 mix
Ethanol	1.0% (v/v)	100	0	0
CP	10.0	--	44	44
Test substance	3	100	2	2
	10	94	2	2
	33	97	1	0

MMC: Mitomycin          CP: Cyclophosphamide

                    

Conclusions:

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

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

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:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

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
Precipitation in the exposure medium was seet at concentrations of 100 µg/mL and above.

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; 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; 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.

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Read across justification

No data on the potential for genetic toxicity of Lauryl laurate (CAS 13945-76-1) are available.

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 2306-88-9

The mutagenic potential of Octyl octanoate (CAS 2306-88-9) was tested in a Salmonella typhimurium reverse mutation assay according to OECD guideline 471 (WoE, Ames, 2000). The following strains were used: TA 1535, TA 1537, TA 100, TA 98 and TA 102. Tester strains were incubated with the test substance up to the limit concentration of 5000 µg/plate, with and without the addition of a metabolic activation system. Experiment I was carried out as a plate incorporation assay, while experiments II and III were performed as pre-incubation assays. No cytotoxicity of the test substance was observed. The positive and negative controls were shown to be valid with and without metabolic activation. Due to slightly induced numbers of revertant colonies for strain TA 1535 without metabolic activation in the second experiment, a third experiment was carried out for strain TA 1535 under the same conditions and at concentrations of 333, 1000, 2500 and 5000 µg/plate. No relevant increase in the number of revertant colonies occurred in the repeat experiment and the effect observed in the second experiment was considered to be biologically irrelevant. Thus, the test substance was considered to be not mutagenic in bacterial cells.

CAS 135800-37-2

The mutagenic potential of Fatty acids, C8-16, 2-ethylhexyl esters (CAS 135800-37-2) was assessed in a Salmonella typhimurium reverse mutation assay performed using a protocol similar to OECD guideline 471 and under GLP conditions (WoE, Ames, 1990). Tester strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were incubated with test material concentrations of 8, 40, 200, 100 and 5000 µg/plate in Tween 80/bidest. water with and without the addition of a metabolic activation system. Two independent experiments were performed with triplicates each. No toxicity of the test substance was observed. The positive and negative controls were shown to be valid in the presence and in the absence of metabolic activation. The test substance did not induce an increase in reversions in any of the S. typhimurium strains, with or without metabolic activation. Thus, the test substance was considered to be not mutagenic in bacterial cells.

CAS 95912-86-0

The mutagenic potential of Fatty acids, C8-10, C12-18-alkyl esters (CAS 95912-86-0) was assessed in an Ames assay, performed using a protocol similar to OECD guideline 471 and under GLP conditions (WoE, Ames, 1989). Tester strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were incubated with 5 test substance concentrations of up to 5000 µg/plate in Tween 80/bidest water with and without metabolic activation. Two independent experiments were performed with triplicates each, applying the plate incorporation method. Cytotoxicity of the test substance was observed at 5000 µg/plate. The positive and negative controls were shown to be valid in the presence and in the absence of metabolic activation. The test substance did not induce an increase in reversions in any of the S. typhimurium strains, with or without metabolic activation. Thus, the test substance was considered to be not mutagenic in bacterial cells.

Genetic toxicity (cytogenicity) in mammalian cells in vitro

CAS 26399-02-0

The cytogenetic potential of 2-ethylhexyl oleate (CAS 26399-02-0) was assessed in an in vitro mammalian chromosome aberration test in primary human lymphocytes, performed according to OECD guideline 473 (key, CA, 2010). Duplicate cultures of human lymphocytes were evaluated for chromosome aberrations in the presence and absence of metabolic activation. In the first experiment, cells were incubated with test substance concentrations of 3, 10 and 33 µg/mL in ethanol for 3 hours with a 24 hours fixation time in the absence and presence of a metabolic activation system. In the second experiment cells were incubated with 3, 10 and 33 µg/mL for 24 hours followed by 24 hours expression time and for 48 hours following 48 hours expression time, all without metabolic activation. In the presence of metabolic activation 2-ethylhexyl oleate was also tested with 3, 10 and 33 µg/mL for 3 hours followed by 48 hours expression time. 33 µg/mL was the maximum concentration due to the limited solubility of the test substance. The highest concentration of the test substance caused modest cytotoxicity. The vehicle (solvent) and positive controls were shown to be valid. The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, with or without metabolic activation. Thus, the test substance was considered to be not clastogenic in mammalian cells.

Genetic toxicity (mutagenicity) in mammalian cells in vitro

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 (key, MLA, 2010). Two independent experiments (with 3 or 24 hours of exposure) were performed in mouse lymphoma L5178Y cells in the absence and presence of metabolic activation (S9-mix) with test substance concentrations up to 100 μg/mL dissolved in ethanol. Precipitation was seen at concentrations of 100 µg/mL and higher. The positive and negative controls were valid and within the range of historical control data. No significant increase in mutation frequency was observed. Thus, the test substance was considered to be not mutagenic in mammalian cells.

Overall conclusion for genetic toxicity

There are no available studies on the genetic toxicity of the target substanceLauryl laurate. Therefore analogue read-across from source substances was applied from in vitro studies on cytogenicity, and in vitro studies on gene mutation in bacterial cells and mammalian cells. The results of the available in vitro studies were consistently negative. Based on the available data and following the analogue approach, absence of mutagenic or clastogenic potential in vitro can be anticipated for Lauryl laurate.

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 to Lauryl laurate (CAS 13945-76-1), 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 analogue read-across approach, the available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.