Lanolin, hydrogenated

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

key study

Study period:

February 25th to March 26th, 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Reliability of original study is 1

Justification for type of information:

Read Across justification is given in section 13 of IUCLID.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

as of May 30th, 2008.

Test guideline 3

Guideline:

other: Japan Regulatory Authorities (METI, MHLW, MAFF) and USA, EPA (TSCA) OPPTS harmonised guidelines

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Alcohols, lanolin

EC Number:

232-430-1

EC Name:

Alcohols, lanolin

Cas Number:

8027-33-6

IUPAC Name:

Lanolin Alcohols

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Remarks:

obtained from University of California, Berkeley on culture discs or from Syngenta CTL, Alderley Edge as frozen vials.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Remarks:

obtained from the British Industrial Biological Research Association

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal preparation (S9-mix)

Test concentrations with justification for top dose:

50, 150, 500, 1500 and 5000 mg/plate (Experiment 1 and Experiment 2)
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 mg/plate.

Vehicle / solvent:

- Vehicle used: acetone.
- Justification for choice of vehicle: the test material was insoluble in DMSO at 50 mg/ml but was fully soluble in acetone in solubility checks. Acetone was therefore selected as the vehicle. Sterile distilled water was not selected as a potential vehicle following information supplied by the sponsor.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene 1 μg/plate for TA100, 2 μg/plate for TA1535 and TA1537, 10 μg/plate for WP2urvA

Remarks:

with metabolic activation

Details on test system and experimental conditions:

PREPARATION OF TEST MATERIAL: the test material was accurately weighed and approximate half-log dilutions prepared in acetone by mixing on a vortex mixer and sonication for 1 minute at 40 °C on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity content (10 %) of the test material. Prior to use, the solvent was dried to remove water using molecular sieves ie 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 0.0004 microns.

PREPARATION OF S9 MIX: it was prepared immediately before use using sterilised co-factors and maintened on ice for the duration of the test. 5.0 ml S9, 1.0 ml 1.65M KCl/0.4 M MgCl2 2.5 ml 0.1 M glucose-6-phosphate, 2.0 ml 0.1 M NADP, 25.0 ml 0.2 M sodium phosphate buffer (pH 7.4), 14.5 ml sterile distilled water.

PREPARATION OF AGAR: a 0.5 ml aliquot of S9 mix and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment. Top agar was prepared using 0.6 % Bacto agar (lot no 9070630 01/14) and 0.5 % sodium chloride with 5 ml of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 ml of top agar. Vogel-Bonner Mininimal agar plated were purchased from ILS ltd (lot no 1098680 08/14 and 1107252 10/14).

PRELIMINARY TOXICITY TEST
- Concentrations: 0 (vehicle), 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.
- 0.1 ml of bacterial culture (TA100 or WP2uvrA-) was mixed with 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 plate of Vogel-Bonner Minimal agar (30 ml/plate). In addition, 0.1 ml of the maxiumum concentration of the test material and 2 ml of molten, trace histidine or tryptophan suplpemented, top agar were overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material.
- Incubation conditions: 48 hours, at 37 °C.
- Evaluation of the frequency of revertant colonies: by the use of a Domino colony counter and examined for effects on the growth of the bacterial background lawn. Manual counts were performed at 5000 μg/plate because of test material precipitation.

MUTATION TEST-EXPERIMENT 1
- Method of application: direct plate incorporation.
- Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate).
- Replications: three for each bacterial strain and for each concentration of test material both with and without S9-mix.
- Incubation conditions: at 37 °C, for approximately 48 hours.
- Evaluation of the frequency of revertant colonies: by the use of a Domino colony counter. Manual counts were performed at 5000 μg/plate because of test material precipitation.

MUTATION TEST-EXPERIMENT 2
- Method of application: pre-incubation.
- Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 ml of S9-mix or phosphate buffer and 0.05 ml of the vehicle or test material formulation and incubated for 20 minutes at 37 °C with shaking at approximately 130 rpm prior to addition of 2 ml of molten, trace histidine or tryptophan supplemented, top agar. The contents fo the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate).
- Replications: three for each bacterial strain and for each concentration of test material both with and without S9-mix.
- Incubation conditions: at 37 °C, for approximately 48 hours.
- Evaluation of the frequency of revertant colonies: by the use of a Domino colony counter. Manual counts were performed at and above 1500 μg/plate because of test material precipitation.

Evaluation criteria:

Criteria for determing a positive result: a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolical activation. Biological relevance will be considered first, statistical methods, as reccomended by the UKEMS (1) can also be used as an aid to evaluation, however, statistical significance will not be the only determing factor for a positive response. A test material not meeting the above cirteria will be considered non-mutagenic (negative).

(1) Kirklan, D.J. (ed). 1989. Statistical evaluation of mutagenicity test data UKEMS sub-commitee on guidelines for mutaginicity testing. Report Part III-Cambridge University Press.

Results and discussion

Test results

Test results 1

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:

valid

Positive controls validity:

valid

Test results 2

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
- Precipitation: precipitation was noted at and above 1500 μg/plate in the preliminary toxicity test in the two strains tested (TA100 and WP2uvrA-) both with and without metabolic activation. A greasy, particulate precipitate was noted at and above 1500 and 5000 μg/plate in Experiments 1 and 2, respectively. This observation did not prevent the scoring of revertant colonies.

PRELIMINARY TOXICITY TEST: the test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

STRAIN CHARACTERISTICS: prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate. They were all found to be satisfactory. The amino acid supplemented top agar and S9-mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable.

HISTORICAL CONTROL DATA
- Positive historical control data: the induced marked increases in the frequency of revertant colonies confirm the activity of the S9-mix and the sensitivity of the bacterial strains. 2008: TA100(-S9): 227-1329, TA100(+S9): 303-2854, TA1535(-S9): 79-653, TA1535(+S9): 103-777, TA102(-S9): 719-2525, TA102(+S9): 654-1758, TA98(-S9): 75-703, TA98(+S9): 120-2177, TA1537(-S9): 78-2917, TA1537(+S9): 92-779, TA1538(-S9): 64-1746, TA1538(+S9): 112-1351, WP2uvrA-(-S9):101-1881, WP2uvrA-(+S9): 138-3496, WP2uvrA- pKM101(-S9):432-2786, WP2uvrA- pKM101(+S9): 749-3551, WP2- pKM101(-S9): 431-2571, WP2pKM101(+S9): 206-1465.
2009: TA100(-S9): 235-1633, TA100(+S9): 354-3672, TA1535(-S9): 100-1855, TA1535(+S9): 68-1200, TA102(-S9): 699-2718, TA102(+S9): 522-1555, TA98(-S9): 83-980, TA98(+S9): 105-697, TA1537(-S9):101-2744, TA1537(+S9): 136-626, WP2uvrA-(-S9):95-2474, WP2uvrA-(+S9):167-2191, WP2uvrA- pKM101(-S9):536-4043, WP2uvrA- pKM101(+S9): 800-2875, WP2- pKM101(-S9): 239-4472, WP2pKM101(+S9): 243-3153.
- Negative (solvent/vehicle) historical control data: spontaneous mutation rates were acceptable. 2008: TA100(-S9): 63.-171, TA100(+S9): 62-184, TA1535(-S9): 10-38, TA1535(+S9): 8-36, TA102(-S9): 181-363, TA102(+S9): 204-387, TA98(-S9): 10-50, TA98(+S9): 11-44, TA1537(-S9): 2-27, TA1537(+S9): 4-28, TA1538(-S9): 5-20, TA1538(+S9): 11-22, WP2uvrA-(-S9):12-49, WP2uvrA-(+S9): 12-55, WP2uvrA- pKM101(-S9):82-250, WP2uvrA- pKM101(+S9): 105-271, WP2- pKM101(-S9): 51-147, WP2pKM101(+S9): 63-180.
2009: TA100(-S9): 61-160, TA100(+S9): 62-152, TA1535(-S9): 10-38, TA1535(+S9): 8-39, TA102(-S9): 181-322, TA102(+S9): 215-361, TA98(-S9): 12-39, TA98(+S9): 12-42, TA1537(-S9):2-26, TA1537(+S9): 6-23, WP2uvrA-(-S9):12-52, WP2uvrA-(+S9):16-53, WP2uvrA- pKM101(-S9):91-191, WP2uvrA- pKM101(+S9): 93-250, WP2- pKM101(-S9): 72-152, WP2pKM101(+S9): 89-187.

Any other information on results incl. tables

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.

Table: test results of experiment 1- Without metabolic activation

Test Results: Experiment 1 - Without metabolic activation
Test period		Number of revertants (mean number of colonies per plate)
With or without S9-mix	Test substance concentration (μg/plate)	Base-pair substitution type						Frameshift type
		TA100		TA1535		WP2uvrA-		TA98		TA1537
-	0	86	(89) 7.6#	16	(19) 2.6	22	(21) 1.7	22	(19) 3.0	12	(11) 1.0
		84		21		22		16		10
		98		20		19		19		11
-	50	73	(74) 0.6	22	(20) 3.5	22	(23) 2.1	20	(18) 3.8	14	(12) 2.5
		74		16		21		14		12
		74		22		25		21		9
-	150	82	(81) 9.5	13	(14) 1.7	23	(23) 2.0	21	(20) 1.5	15	(12) 3.0
		71		13		25		20		12
		90		16		21		18		9
-	500	70	(80) 17.0	24	(21) 3.1	22	(19) 3.0	18	(16) 2.1	9	(10) 1.7
		100		18		16		14		12
		71		22		19		15		9
-	1500	90P	(101) 15.9	23P	(21) 1.5	15P	(15) 0.6	15P	(18) 2.3	10P	(12) 2.9
		93P		21P		16P		19P		10P
		119P		20P		15P		19P		15P
-	5000	93P	(92) 1.2	23P	(21) 1.5	18P	(18) 0.6	18P	(18) 0.6	14P	(13) 1.5
		91P		20P		18P		19P		13P
		93P		21P		17P		18P		11P
Positive controls S9-mix (-)	Name	ENNG		ENNG		ENNG		4NQO		9AA
	Conc (μg/plate)	3		5		2		0.2		80
	No colonies per plate	220	(260) 58.2	624	(696) 82.4	386	(415) 26.4	157	(166) 10.1	764	(823) 167.5
		234		679		423		165		693
		327		786		437		177		1012

Table: test results of experiment 1- With metabolic activation

Test period		95
With or without S9-mix	Test substance concentration (μg/plate)	Base-pair substitution type						Frameshift type
		TA100		TA1535		WP2uvrA-		TA98		TA1537
+	0	95	(89) 4.9#	9	(12) 3.0	19	(26) 2.6	22	(21) 15	13	(12) 1.5
		87		12		24		21		10
		86		15		25		19		12
+	50	100	(86) 12.7	13	(11) 1.5	21	(20) 1.2	21	(22) 1.7	12	(10) 2.1
		81		10		21		24		8
		76		11		19		21		11
+	150	87	(79) 9.2	12	(10) 2.0	27	(28) 3.2	21	(20) 5.6	9	(9) 0.6
		81		10		26		25		10
		69		8		32		14		9
+	500	91	(94) 5.2	16	(11) 4.2	23	(21) 2.1	20	(18) 2.9	11	(9) 1.5
		91		10		19		20		8
		100		8		22		15		9
+	1500	90P	(91) 5.0	11P	(10) 2.3	21P	(23) 3.2	19P	(22) 2.6	8P	(10) 1.7
		90P		7P		27P		24P		11P
		86P		11P		22P		23P		11P
+	5000	96P	(91) 4.6	10P	(10) 0.6	26P	(23) 2.5	20P	(21) 1.0	12P	(11) 1.2
		87P		11P		23P		21P		10P
		90P		10P		21P		22P		12P
Positive controls S9-mix (+)	Name	2AA		2AA		2AA		BP		2AA
	Conc (μg/plate)	1		2		10		5		2
	No colonies per plate	317	(312) 4.6	161	(186) 26.0	588	(531) 75.2	231	(275) 38.9	109	(127) 19.8
		311		185		446		305		123
		308		213		560		289		148

Table: test results of experiment 2- Without metabolic activation

Test period		Number of revertants (mean number of colonies per plate)
With or without S9-mix	Test substance concentration (μg/plate)	Base-pair substitution type						Frameshift type
		TA100		TA1535		WP2uvrA-		TA98		TA1537
-	0	93	(106) 13.0#	21	(18) 3.1	12	(14) 2.1	16	(18) 5.7	12	(10) 1.5
		119		17		13		24		10
		107		15		16		13		9
-	50	127	(109) 16.3	21	(17) 3.2	18	(19) 5.6	25	(22) 3.0	16	(11) 5.0
		106		16		25		22		11
		95		15		14		19		6
-	150	99	(109) 9.1	25	(24) 6.1	15	(23) 7.0	15	(21) 7.9	13	(11) 2.9
		116		17		28		30		13
		113		29		26		18		8
-	500	124P	(117) 8.9	31P	(23) 7.5	28P	(20) 6.8	31P	(22) 8.5	12P	(11) 1.7
		132P		16P		15P		14P		12P
		96P		22P		18P		21P		9P
-	1500	88P	(94) 5.7	17P	(18) 3.2	23P	(20) 2.6	12P	(12) 0.0	9P	(10) 2.1
		99P		22P		18P		12P		8P
		96P		16P		19P		12P		12P
-	5000	106P	(104) 7.2	28P	(21) 5.9	22P	(23) 4.0	14P	(14) 3.5	6P	(9) 3.0
		96P		17P		27P		10P		12P
		110P		19P		19P		17P		9P
Positive controls S9-mix (-)	Name	ENNG		ENNG		ENNG		4NQO		9AA
	Conc (μg/plate)	3		5		2		0.2		80
	No colonies per plate	278	(321) 37.0	107	(108) 4.6	348	(350) 27.5	130	(124) 10.1	1247	(1820) 496.9
		340		104		323		129		2136
		344		113		378		112		2076

Table: test results of experiment 2- With metabolic activation

Test period		Number of revertants (mean number of colonies per plate)
With or without S9-mix	Test substance concentration (μg/plate)	Base-pair substitution type						Frameshift type
		TA100		TA1535		WP2uvrA-		TA98		TA1537
+	0	121	(110) 9.9#	18	(13) 4.0	28	(24) 8.4	28	(25) 11.2	13	(16) 3.5
		103		11		29		13		20
		105		11		14		35		16
+	50	86	(98) 11.0	13	(12) 1.0	30	(26) 6.1	18	(18) 2.5	10	(15) 6.1
		102		11		29		20		14
		107		12		19		15		22
+	150	114	(114) 1.5	8	(11) 4.4	22	(24) 3.8	26	(24) 7.2	19	(15) 3.5
		116		9		28		16		15
		113		16		21		30		12
+	500	104P	(114) 10.0	9P	(14) 4.6	15P	(23) 9.2	19P	(26) 5.9	14P	(17) 2.9
		124P		15P		33P		28P		19P
		114P		18P		21P		30P		19P
+	1500	111P	(105) 16.8	10P	(10) 2.0	28P	(28) 2.5	20P	(24) 4.0	10P	(12) 1.5
		86P		12P		30P		28P		13P
		118P		8P		25P		25P		12P
+	5000	105P	(107) 2.6	9P	(11) 2.6	30P	(26) 4.5	23P	(21) 2.6	8P	(13) 5.0
		110P		10P		21P		18P		14P
		106P		14P		26P		22P		18P
Positive controls S9-mix (+)	Name	2AA		2AA		2AA		BP		2AA
	Conc (μg/plate)	1		2		10		5		2
	No colonies per plate	715	(755) 35.8	18	(176) 16.9	290	(297) 12,7	282	(287) 4.6	126	(123) 3.8
		784		157		290		288		119
		766		190		312		291		125

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4 -Nitroquinoline-1 -oxide

9AA: 9 -Aminoacridine

2AA: 2 -Aminoanthracene

BP: Benzopyrene

P: Precipitate

#: Standard deviation

Applicant's summary and conclusion

Conclusions:

The substance is not mutagenic under tested conditions.

Executive summary:

The test material was tested for its mutagenic potential in the Ames test, according to the OECD Guideline 471, EU Method B13/14, Japan Regulatory Authorities and USA, EPA OPPTS harmonised guidelines. A preliminary toxicity test (range was performed by using TA100 and WP2uvrA bacterial strains tested in 10 concentrations ranging from 0.15 to 5000 μg/plate. The test material was non-toxic to the strains of bacteria used. Subsequently, two individual experiments were performed by the use of direct plate incorporation and pre-incubation method. S.Triphimurium strains TA1535, TA1537,TA98, TA100 and E.coli WP2uvrA were treated with the test material using both Ames plate incorporation and pre-incubation methods 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 experiment was repeated on a separate day by using the pre-incubation method and by using fresh cultures of the bacterial strains and fresh test formulations. Negative (vehicle control-acetone) and positive controls run in parallel.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 mg/plate. A greasy, particulate precipitate was noted at and above 1500 and 5000 μg/plate in Experiments 1 and 2, respectively. This observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.

The test material is considered as non-mutagenic under tested conditions.