Lanolin, acetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 08 March 2010 and 07 June 2010.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study from supporting substance (structural analogue)

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 15th September 2009 Date of signature: 26th November 2009

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Lanolin Alcohols

IUPAC Name:

Lanolin Alcohols

Method

Target gene:

The L5178Y TK+/- locus of the L5178Y mouse lymphoma cell line

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 µg/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/ml) and 10% donor horse serum (giving R10 media) at 37°C with 5% CO2 in air.

- Properly maintained: yes

- Periodically checked for Mycoplasma contamination: yes

- Periodically checked for karyotype stability: yes

- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/ beta-naphthaflavone

Test concentrations with justification for top dose:

The dose range used in the preliminary toxicity test was 9.77 to 2500 µg/ml for all three of the exposure groups.

Main experiment - 4 hours with and without S9: 0, 39.06, 78.13, 156.25, 312.5, 625 and 937.5 µg/ml
Main experiment - 24 hours without S9: 0, 39.06, 78.13, 156.25, 208.33, 260.41 and 312.5 µg/ml

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: not stated in report

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (acetone) treatment groups were used as the vehicle control

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Used at 400 µg/ml and 150 µg/ml for the 4 and 24 hours exposures respectively, in absence of S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (acetone) treatment groups were used as the vehicle control

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Used at 2 µg/ml in presence of S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours (with S9) and 24 hours (without S9)
- Expression time (cells in growth medium): 2-day expression period

SELECTION AGENT (mutation assays): TFT = Trifluorothymidine Deoxyriboside

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: The cells were counted, diluted to 104 cells/ml and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/ml 5 trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/ml and plated (2 cells/well) for viability (%V) in non-selective medium.

DETERMINATION OF CYTOTOXICITY
- Method: Relative suspension growth (RSG), induced mutant frequency (IMF), gloval evaluation factor (GEF)

OTHER EXAMINATIONS:
- None

Evaluation criteria:

Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.

Dose levels that have survival values less than 10% are usually excluded from any statistical analysis, as any response they give would be considered to have no biological or toxicological relevance.

For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. Following discussions at an International Workshop on Genotoxicity Test Procedures in Plymouth, UK, 2002 (Moore et al 2003) it was felt that the IMF must exceed some value based on the global background MF for each method (agar or microwell). This Global Evaluation Factor (GEF) value was set following a further meeting of the International Workshop in Aberdeen, Scotland, 2003 (Moore et al 2006) at 126 x 10-6 for the microwell method. Therefore any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10-6 will be considered positive. However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test material induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.

Statistics:

Small significant increases designated by the UKEMS statistical package will be reviewed using the above criteria, and may be disregarded at the Study Director's discretion.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

39.06 µg/ml and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no marked change in pH when the test material was dosed into media
- Effects of osmolality: osmolality did not increase by more than 50 mOsm.
- Evaporation from medium: not applicable to this test substance
- Water solubility: not soluble in water
- Precipitation: Increased levels of precipitation reduced exposure to the cells.
- Other confounding effects: none stated

RANGE-FINDING/SCREENING STUDIES: In all three of the exposure groups there was a marked reduction in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. Precipitate of the test material was observed at and above 19.53 µg/ml, which was seen to aggregate at and above 625 µg/ml and effectively reduce exposure to the cells. This is supported by the fact that maximum toxicity in both of the 4-hour exposure groups was achieved at 625 µg/ml. In the subsequent mutagenicity test the maximum dose was limited by the estimated maximum test material exposure and induced toxicity.

COMPARISON WITH HISTORICAL CONTROL DATA: Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary toxicity test

The dose range of the test material used in the preliminary toxicity test was 9.77 to 2500 µg/ml. The results for the Relative Suspension Growth (%) were as follows:

Dose (µg/ml)	% RSG (-S9) 4-Hour Exposure	% RSG (+S9) 4-Hour Exposure	% RSG (-S9) 24-Hour Exposure
0	100	100	100
9.77	103	118	110
19.53	98	92	145
39.06	84	112	132
78.13	93	79	104
156.25	65	74	73
312.5	50	62	4
625	38	50	0
1250	88	91	0
2500	91	105	10

4 -Hour Exposure With and Without Metabolic Activation

There was evidence of toxicity following exposure to the test material in both the presence and absence of metabolic activation, as indicated by the %and RTG values. There was no evidence of a reduction in viability (%V), therefore indicating that no residual toxicity occurred, in both the absence and presence of metabolic activation. Optimum levels of toxicity were not achieved in the absence of metabolic activation with a maximum of 55% (RTG) being observed at 625 µg/ml, and 21% toxicity at the higher dose of 937.5 µg/ml. This confirmed the observation in the preliminary toxicity test that maximum exposure was achieved at 625 µg/ml. In the presence of metabolic activation optimum levels of toxicity were observed at 312.5 µg/ml, whilst less toxicity was observed at higher dose levels again confirming that increased levels of precipitate were reducing exposure to the cells. It was considered that the test material had been adequately tested. Acceptable levels of toxicity were seen with both positive control substances.

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6per viable cell in both the presence and absence of metabolic activation. In both of the 4-hour exposure groups, some of the test material dose levels had mutant frequencies greater than the vehicle control value. However, there were no clear dose-related responses and all the values were within the acceptable range for vehicle controls and, therefore, they were considered to be of no toxicological significance.

24-Hour Exposure Without Metabolic Activation

As was seen previously, there was evidence of a marked reduction in %and RTG values in cultures dosed with the test material . There was also evidence of modest reductions in viability (%V), therefore indicating that modest residual toxicity had occurred. Optimum levels of test material-induced toxicity were achieved at 208.33 µg/ml.  The positive control induced acceptable levels of toxicity.

The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had a modest effect on the toxicity of the test material.

The vehicle control mutant frequency value was within the acceptable range of 50 to 200 x 10^-6viable cells. The positive control produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily.

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6per viable cell. As was seen previously, any increases in mutant frequency over the controls were small, were within the acceptable range for vehicle controls and were therefore considered to be of no toxicological significance (Table 9). A precipitate of test material was observed at and above 78.13 µg/ml.

Applicant's summary and conclusion

Conclusions:

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Executive summary:

Introduction. This study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476) and Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008..

Methods. One main experiment was performed. In this main experiment, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at six levels, in duplicate, together with vehicle (acetone) and positive controls. The exposure groups used were as follows: 4‑hour exposures both with and without metabolic activation, and 24–hour exposure without metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test and was 39.06 to 937.5µg/ml for the 4 -hour exposure groups in the presence and absence of metabolic activation, and 39.06 to 312.5µg/ml for the 24 hour exposure in the absence of metabolic activation.

Results. The maximum dose level used in the mutagenicity test was limited by the estimated maximum exposure to the test material and test material induced toxicity. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any toxicologically significant or dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in any of the three exposure groups.

Conclusion. The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.