Reaction mass of 2-ethylhexyl 12-[(12-hydroxyoctadecanoyl)oxy]octadecanoate and 2-ethylhexyl 12-hydroxyoctadecanoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 October 2007 to 12 October 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

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

Test material

Test material information

1

Reference substance name:

2-ethylhexyl 12-hydroxyoctadecanoate

EC Number:

249-793-7

EC Name:

2-ethylhexyl 12-hydroxyoctadecanoate

Cas Number:

29710-25-6

Molecular formula:

C26H52O3

IUPAC Name:

2-ethylhexyl 12-hydroxyoctadecanoate

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: University of California at Berkeley on culture discs on 4 August 1995
- Suitability of cells: Yes
- Methods for maintenance in cell culture if applicable: All of the strains were stored at
-196°C in a Statebourne liquid nitrogen freezer. Prior to the master strains being
used, characterization checks were carried out to confirm the amino-acid requirement, presence of
rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate. Overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37 deg C for 10 hours.
- Normal (negative control) cell cycle time: Yes

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: plate method
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: Not reported
- Periodically checked for karyotype stability: Not reported
- Periodically 'cleansed' against high spontaneous background: Yes

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: British Biological Research Association on 17 August 1987
- Suitability of cells: Yes
- Methods for maintenance in cell culture if applicable: All of the strains were stored at
-196°C in a Statebourne liquid nitrogen freezer. Prior to the master strains being
used, characterization checks were carried out to confirm the amino-acid requirement, presence of
rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate. Overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37 deg C for 10 hours.
- Normal (negative control) cell cycle time: Yes

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: plate method
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: Not reported
- Periodically checked for karyotype stability: Not reported
- Periodically 'cleansed' against high spontaneous background: Yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate metabolising system (10% liver S9 in standard co-factors)

Test concentrations with justification for top dose:

Experiment 1 was determined in a preliminary toxicity assay and was 50, 150, 500, 1500 and 5000 µg/plate. Experiment 2 was performed using the same methology and test concentrations as experiment 1.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test material was immiscible in dimethyl sulphoxide at 50 mg/ml but was fully miscible in acetone at the same concentration in solubility checks performed in-house. Sterile distilled water was not selected as a potential vehicle following information supplied by the sponsor. Acetone was therefore selected as the vehicle.

Controls

Untreated negative controls:

yes

Remarks:

2-Aminoanthracene (2AA) & Benzo(a)pyrene (BP),: 2AA at 1 pg/plate for TAlOO 2AA at 2 pg/plate for TA1535 and TA1537 BP at 5 pg/plate for TA98 2AA at 10 pg/plate for W2uvrA

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Preincubation period: 10 hours
- Exposure duration: 48 hours
- Expression time (cells in growth medium):N/A
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A

SELECTION AGENT (mutation assays): N/A

SPINDLE INHIBITOR (cytogenetic assays): N/A

STAIN (for cytogenetic assays): N/A

NUMBER OF REPLICATIONS: Triple

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: N/A

NUMBER OF CELLS EVALUATED: Not reported

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): N/A

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: N/A

DETERMINATION OF CYTOTOXICITY
- Method: colony
- Any supplementary information relevant to cytotoxicity: N/A

OTHER EXAMINATIONS:
- Determination of polyploidy: N/A
- Determination of endoreplication: N/A
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): N/A

- OTHER: N/A

Evaluation criteria:

There are several criteria for determining a positive results, such as a dose-related increase in revertant frequency over the dose range tested and/or reproductive increase at one or more conc. in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical significance will not ve the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if hte above criteria are not met.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not reported
- Effects of osmolality: Not reported
- Evaporation from medium: Not reported
- Water solubility: Not reported
- Precipitation: Not reported
- Definition of acceptable cells for analysis: Not reported
- Other confounding effects: Not reported

RANGE-FINDING/SCREENING STUDIES: In order to select appropriate dose levels for use in the main test, a preliminary test was carried
out to determine the toxicity of the test material using concentrations of 0,0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 micrograms/plate. The test was performed by mixing 0.1 mL of bacterial culture (TA100 or WP2uvrA-), 2 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of test material formulation and 0.5 mL of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). Ten concentrations of hte test material formulation and a vehicle control (acetone) were tested. In addition, 0.1 mL of the max. concentration of the test material and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onton sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37 deg C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial lawn.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: N/A

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: N/A
- Indication whether binucleate or mononucleate where appropriate: N/A

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Table attached in background information section.
- Negative (solvent/vehicle) historical control data: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are presented in the standard test method section 3 with historical control ranges for 2005 and 2006. Table attached in background information section.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: N/A
- Other observations when applicable: N/A

Any other information on results incl. tables

Table 1: Preliminary toxicity assay: Number of revertant colonies

± S9-mix	Strain	No of Revertant Colonies
		Dose µg/plate
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	65	110	65	78	75	75	105	90	90	79	78P
+	TA100	112	17	94	95	76	85	80	114	91	79	74P
-	WP2uvrA-	25	16	23	19	16	16	14	16	19	14	22P
+	WP2uvrA-	24	16	27	18	32	29	34	27	28	28	20P
P: Precipitate

 

Table 2: Spontaneous Mutation Rates (Concurrent Negative Controls)

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA-		TA98		TA1537
Experiment 1
76	(68)	16	(11)	22	(21)	10	(13)	2	(6)
65		8		14		14		8
63		10		27		14		9
Experiment 2
91	(107)	25	(27)	34	(40)	20	(21)	17	(16)
131		28		40		24		14
99		27		46		18		17

 

Table 3: Results – Experiment 1

± S9-Mix	Test substance concentration (µg/plate)	Number of Revertants (mean number of colonies per plate)
		Base-pair substitution type						Frameshift type
		TA100		TA1535		WP2uvrA-		TA98		TA1537
-	0	79	(79) 1.0#	10	(10) 0.6	22	(26) 8.1	16	(16) 2.0	9	(9) 0.0
		78		10		20		18		9
		80		11		35		14		9
-	50	73	(76) 3.1	10	(12) 2.6	22	(25) 9.5	12	(12) 2.0	9	(8) 1.2
		75		11		18		10		9
		79		15		36		14		7
-	150	93	(86) 14.5	14	(13) 1.0	16	(25) 9.0	12	(12) 1.0	12	(10) 2.1
		69		13		26		11		9
		95		12		34		13		8
-	500	78	(76) 2.6	11	(11) 4.5	18	(18) 0.6	15	(13) 1.7	3	(5) 2.0
		77		16		19		12		7
		73		7		18		12		5
-	1500	78P	(76) 5.7	7P	(6) 1.2	23P	(24) 2.6	11P	(13) 2.1	13P	(13) 2.5
		70P		5P		23P		15P		10P
		81P		5P		27P		12P		15P
-	5000	79P	(76) 6.4	8P	(8) 2.5	29P	(26) 2.6	18P	(16) 2.0	8P	(10) 2.6
		69P		5P		24P		16P		13P
		81P		10P		25P		14P		9P
Positive controls (- S9-Mix)	Name	ENNG		ENNG		ENNG		4NQO		9AA
	Concentration (µg/plate)	3		5		2		0.2		80
	No. colonies per plate	750	(690) 85.4	362	(400) 33.0	1202	(1166) 50.9	115	(117) 4.9	2754	(2817) 67.4
		727		422		1108		114		2808
		592		416		1189		123		2888
+	0	75	(73) 2.1#	10	(10) 1.5	29	(31) 2.1	12	(18) 5.6	8	(7) 1.7
		74		8		33		19		5
		71		11		30		23		8
+	50	71	(71) 0.6	10	(11) 0.6	33	(28) 5.0	20	(18) 4.7	2	(2) 2.0
		70		11		23		22		2
		71		11		29		13		2
+	150	73	(70) 5.2	11	(10) 1.5	38	(29) 7.9	23	(23) 7.0	8	(7) 5.0
		73		10		26		30		2
		64		8		23		16		12
+	500	90	(76) 15.0	10	(9) 1.2	25	(24) 3.1	12	(15) 2.3	3	(5) 4.9
		77		8		21		16		11
		60		10		27		16		2
+	1500	74P	(69) 5.0	11P	(9) 1.5	27P	(27) 0.6	25P	(20) 5.0	5P	(6) 1.2
		64P		9P		26P		15P		7P
		69P		8P		27P		21P		7P
+	5000	78P	(70) 6.8	2P	(5) 3.5	26P	(25) 0.6	13P	(14) 3.6	7P	(7) 0.0
		68P		5P		25P		11P		7P
		65P		9P		25P		18P		7P
Positive controls (+ S9-Mix)	Name	2AA		2AA		2AA		BP		2AA
	Concentration (µg/plate)	1		2		10		5		2
	No. colonies per plate	509	(440) 60.0	128	(139) 49.9	390	(416) 97.6	229	(240) 28.5	296	(244) 47.3
		398		95		334		272		204
		414		193		524		218		231
ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine 4NQO: 4-Nitronquinoline-1-oxide 9AA: 9-Aminoacridine P: Precipitate BP: Benzo(a)pyrene # Standard Deviation

 

Table 4: Results - Experiment 2

± S9-Mix	Test substance concentration (µg/plate)	Number of Revertants (mean number of colonies per plate)
		Base-pair substitution type						Frameshift type
		TA100		TA1535		WP2uvrA-		TA98		TA1537
-	0	106	(98) 6.8#	25	(26) 3.1	35	(36) 3.6	24	(23) 3.2	18	(16) 2.1
		93		29		33		25		14
		96		23		40		19		15
-	50	96	(84) 10.1	22	(21) 1.7	34	(34) 0.0	16	(17) 1.2	19	(19) 0.6
		79		22		34		18		18
		78		19		34		16		19
-	150	90	(92) 2.5	18	(23) 6.4	30	(31) 2.3	15	(15) 5.5	20	(16) 4.5)
		95		20		30		21		16
		92		30		34		10		11
-	500	93	(93) 2.5	15	(19) 3.6	30	(32) 2.1	14	(16) 3.2	25	(18) 7.0
		95		20		34		15		19
		90		22		33		20		11
-	1500	90P	(93) 3.6	18P	(25) 7.5	34P	(34) 0.6	26P	(18) 8.0	9P	(13) 3.2
		92P		33P		34P		18P		15P
		97P		24P		35P		10P		14P
-	5000	92P	(91) 1.0	26P	(26) 0.6	33P	(34) 1.5	12P	(19) 6.1	8P	(14) 6.0
		91P		27P		34P		23P		15P
		90P		26P		36P		22P		20P
Positive controls (- S9-Mix)	Name	ENNG		ENNG		ENNG		4NQO		9AA
	Concentration (µg/plate)	3		5		2		0.2		80
	No. colonies per plate	1028	(969) 52.8	749	(681) 88.3	1257	(1235) 23.0	243	(206) 41.0	1572	(1255) 304.0
		926		581		1211		214		966
		953		712		1236		162		1227
+	0	97	(110) 11.2#	15	(12) 3.0	44	(44) 0.6	25	(26) 2.6	18	(18) 3.0
		118		12		44		29		21
		114		9		45		25		15
+	50	81	(87) 5.6)	14	(12) 2.5	42	(41) 2.3	24	(24) 2.0	14	(17) 3.6
		88		9		42		22		16
		92		12		38		26		21
+	150	100	(100) 11.0	12	(12) 0.6	37	(37) 0.6	25	(22) 6.1	16	(17) 3.1
		111		12		37		15		14
		89		13		36		26		20
+	500	93	(92) 9.1	15	(13) 2.1	37	(40) 3.1	22	(21) 0.6	13	(13) 0.6
		100		11		43		21		12
		82		12		41		21		13
+	1500	91P	(101) 10.6	5P	(8) 2.6	37P	(37) 0.0	23P	(23) 0.6	11P	(13) 2.1
		99P		10P		37P		23P		15P
		112P		9P		37P		22P		14P
+	5000	110P	(102) 13.6	16P	(13) 2.5	42P	(41) 3.6	18P	(18) 0.6	12P	(12) 3.5
		86P		11P		44P		19P		15P
		109P		13P		37P		18P		8P
Positive controls (+ S9-Mix)	Name	2AA		2AA		2AA		BP		2AA
	Concentration (µg/plate)	1		2		10		5		2
	No. colonies per plate	891	(880) 10.5	198	(193) 8.1	299	(326) 37.6	250	(274) 23.1	364	(375) 56.9
		870		198		310		277		325
		879		184		369		296		437
ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine 4NQO: 4-Nitronquinoline-1-oxide 9AA: 9-Aminoacridine P: Precipitate BP: Benzo(a)pyrene # Standard Deviation

 

Applicant's summary and conclusion

Conclusions:

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activition. The test material was considered non-mutagenic under the conditions of this study.

Executive summary:

The study was conducted according to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the Experiment 1 was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. the test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A Precipitate (oily in appearance) was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activition. The test material was considered non-mutagenic under the conditions of this study.