Reaction mass of Fatty acids, montan-wax and Fatty acids, montan-wax, ethylene esters and Fatty acids, montan wax, mixed esters with fatty acids C16-18 and ethylene glycol and Fatty acids, montan-wax, stearyl esters and Montan wax

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Studies (RL1) are available, which investigated potential mutagenic effects in bacteria (Montan waxes, types WM 31 and NaV 101, WE 4, E and LG), chromosomal aberrations (Montan waxes, types S, NaV 101, O, WM 31 and LG) and gene mutations in mammalian cells (Montan waxes, types S, NaV 101, O, WM 31 and LG). These in vitro studies with different types of Montan waxes did not observe genotoxic effects.

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:

From 20 June 2017 to 31 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 liver extract from phenobarbital (PB) and β-naphthoflavone (BNF) induced male Wistar rats

Test concentrations with justification for top dose:

Pre-Test: 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate
Main study: 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate, additionally 1.581 µg/plate in complementary confirmatory tests
Top dose according to guideline

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: polyethylene glycol PEG 400

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylenediamine (TA98), Sodium azide (TA100, TA1535), 9-aminoacridine (TA1537), Methyl-methanesulfonate (WP2 uvrA)

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (all strains)

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) in initial mutation test, preincubation in confirmatory mutation test and complementary confirmatory mutation test

DURATION
- Preincubation period: 20 min at 37 °C
- Exposure duration: 48 +/- 1 h
- Fixation time (start of exposure up to fixation or harvest of cells): 48 +/- 1 h

NUMBER OF REPLICATIONS: triplicates for each dose level

DETERMINATION OF CYTOTOXICITY
- Method: other: number of revertant colonies and inhibition of background lawn

Evaluation criteria:

A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- the number of reversions was more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and
Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions was more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

The test item was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

not performed

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: not in initial test, but in confirmatory test without metabolic activation at >/= 1581 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

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 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: not in initial test, but in confirmatory test without metabolic activation at >/= 1581 µg/plate µg/plate, with metabolic activation at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium 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

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:

not examined

Positive controls validity:

valid

Additional information on results:

Summary tables are attached to his study record

Slight precipitation or precipitation of the test item was observed at concentrations >/= 500 µg/plate.

Conclusions:

Under the conditions of this study, the test item was not mutagenic to bacteria.

Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method), a Confirmatory Mutation Test (Pre-Incubation Method). Due to excessive cytotoxicity in parts of the study by the Pre-Incubation Method, a Complementary Confirmatory Test was performed by the Plate Incorporation (Salmonella typhimurium TA100 and TA1537 without metabolic activation) or Pre-Incubation Method (Salmonella typhimurium TA1535 without metabolic activation).

Based on the results of the Compatibility Test, the test item was dissolved in PEG 400 at a concentration of 100 mg/mL. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test in tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation.

Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test and Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50 15.81 and 5 μg/plate; in the Complementary Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate in case of Salmonella typhimurium TA100 and TA1537 without metabolic activation and 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate in case of Salmonella typhimurium TA1535 without metabolic activation.

Precipitate/slight precipitate was detected on the plates in the Preliminary Concentration Range Finding Test at higher concentrations with and without metabolic activation in all examined bacterial strains.

Precipitate/slight precipitate/ was detected on the plates in the main tests at higher concentrations with and without metabolic activation in all examined bacterial strains with the exception of Escherichia coli WP2 uvrA strain with and without metabolic activation in the Initial Mutation Test. Inhibitory, cytotoxic effect of the test item was not detected in the Preliminary Concentration Range Finding Test and in the Initial Mutation Test. In the Confirmatory Mutation Test slightly reduced background lawn or lower numbers of revertant colonies was detected in Salmonella typhimurium TA98 strain with and without metabolic activation at higher concentrations. In the Complementary Confirmatory Mutation Test slightly reduced background lawn was observed in Salmonella typhimurium TA1535 strain without metabolic activation at higher concentrations.

In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any treatment-related effect. No historical control database exists for PEG 400; however, the observed data for this solvent was confirmed to be comparable with the historical control range of other solvents, therefore it was considered to be acceptable. Some cytotoxicity was observed with the vehicle under some conditions, but sufficient data was generated within the study for the results to be fully valid.

The mean values of revertant colonies of the other solvent control plates were in harmony the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests. The tests were considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used .

In conclusion, the test item WARADUR LG (Batch Number: W00004534) has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 28 June 2017 to 6 June 2018

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)

Version / remarks:

29 July 2016

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 liver extract from phenobarbital (PB) and β-naphthoflavone (BNF) induced male Wistar rats

Test concentrations with justification for top dose:

0, 0.41; 1.23; 3.70; 11.11; 33.33; 100; 300 µg/mL in all experiments
based on a preliminary cytotoxicity test with concentrations up to 2000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: propylene glycol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 5 x 10exp5 per dish

DURATION
- Exposure duration: 3 h (experiments 1 and 2 with metabolic activation, experiment 1 without metabolic activation), or 20 h (experiment 2 and 3 without metabolic ativation)
- Expression time (cells in growth medium): 17 h for 3 h exposures
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h (all experiments)

SPINDLE INHIBITOR (cytogenetic assays): Colchicine

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates for each dose level

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): at least 150 metaphases with 22 +/- 2 chromosomes from each culture, i.e. 300 per dose level

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

The test item is considered to have shown clastogenic activity in this study if all of the following criteria are met:
- Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps will be considered).
- The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
- The increases are statistically significant.
- The increases are not associated with large changes in pH or osmolality of the treated cultures.
The historical control data for this laboratory were also considered in the evaluation. Evidence of a dose-response relationship (if any) was considered to support the conclusion.
The test item is concluded to have given a negative response if no reproducible, statistically significant increases are observed.

Statistics:

Fisher’s exact test

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: only very slight cytotoxicity (minimum relative gowth: 82 %)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no large changes
- Effects of osmolality: no large changes
- Precipitation: see below
- Definition of acceptable cells for analysis: metaphases with 22 +/- 2 chromosomes

RANGE-FINDING/SCREENING STUDIES:
from 3.096 to 2000 µg/mL

Concentrations scored for chromosomal aberrations:

Experiment 1 (3 h) with and without metabolic activation: 300, 100, 33.33 and 11.11 μg/mL. Insolubility (precipitate, oily film) was detected at the end of the treatment period in the final treatment medium in the 300-11.11 μg/mL concentration range with metabolic activation and in the 300-33.33 μg/mL concentration range without metabolic activation.

Experiment 2 (3 h with metabolic activation): 33.33, 11.11 and 3.70 μg/mL. Insolubility (precipitate, oily film) was detected at the end of the treatment period in the final treatment medium in the 300-3.70 μg/mL concentration range with metabolic activation.

Experiment 2 (20 h without metabolic activation): invalid, repeated (experiment 3)

Experiment 3 (20 h without metabolic activation): 33.33, 11.11 and 3.70 μg/mL. Insolubility (precipitate, oily film) was detected at the end of the treatment period in the final treatment medium in the 300-11.11 μg/mL concentration range without metabolic activation

Conclusions:

Under the conditions of this study, the test item was not clastogenic to mammalian cells in vitro.

Executive summary:

WARADUR LG was tested in vitro in a Chromosome Aberration Assay using Chinese hamster V79 lung cells and according to OECD testing guideline 473. The test item was formulated in Propylene glycol. In independent Chromosome Aberration Assays using duplicate cultures, at least 300 well-spread metaphase cells (or until a clear positive response was detected) were analysed for each evaluated test item treated, negative (vehicle) and positive control sample or untreated control sample in some cases.

In Chromosome Aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 300, 100, 33.33, 11.11, 3.70, 1.23 and 0.41 μg/mL (experiment with and without metabolic activation).

In Assay 1, insolubility was detected at the end of the treatment period in the final treatment medium in the 300-11.11 μg/mL concentration range with metabolic activation and in the 300-33.33 μg/mL concentration range without metabolic activation. There were no large changes in the pH or osmolality with and without metabolic activation. No marked cytotoxicity was observed in any samples of this assay. Therefore, concentrations of 300, 100, 33.33 and 11.11 μg/mL (a total of four) were chosen for evaluation in the experiment with and without metabolic activation.

In Chromosome Aberration Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) was performed. Sampling was performed 20 hours after the beginning of the treatment. The examined concentrations of the test item were 300, 100, 33.33, 11.11, 3.70, 1.23 and 0.41 μg/mL (experiment with metabolic activation).

In Assay 2, insolubility was detected at the end of the treatment period in the final treatment medium in the 300-3.70 μg/mL concentration range with metabolic activation. There were no large changes in the pH and osmolality with metabolic activation. No marked cytotoxicity was observed in any samples of this assay. Therefore, concentrations of 33.33, 11.11 and 3.70 μg/mL (a total of three) were chosen for evaluation in the experiment with metabolic activation.

In Chromosome Aberration Assay 2, a 20-hour treatment without metabolic activation (in the absence of S9-mix) was also performed and scored. Based on the observed results of Assay 2 without metabolic activation

(i.e. high chromosomal aberration frequency of the untreated / negative control samples in Assay 2 without metabolic activation), this Assay was considered invalid and was repeated with identical conditions (see Assay 3).

In Assay 3, insolubility was detected at the end of the treatment period in the final treatment medium in the 300-11.11 μg/mL concentration range without metabolic activation. There were no large changes in the pH and osmolality without metabolic activation. No marked cytotoxicity was observed in any samples of this assay. Therefore, concentrations of 33.33, 11.11 and 3.70 μg/mL (a total of three) were chosen for evaluation in the experiment without metabolic activation.

In summary, none of the treatment concentrations caused a biologically or statistically significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation when compared to the appropriate negative (vehicle) control or untreated control values. The experiments with and without metabolic activation in Assay 1 and with metabolic activation in Assay 2 were clearly negative. Although the data of Assay 3 at 3.70 and 33.33 µg/mL was statistically significant if compared to the negative vehicle control, yet there was no clear dose-response and the increases were not reproducible between replicates. Furthermore, there was no significant increase in the number of aberrant cells at 3.70 and 33.33 μg/mL concentrations if they were compared to the untreated control. Therefore, it was concluded that the test item has not been shown to be clastogenic.

The occurrence of polyploid and endoreduplicated metaphases is documented. Polyploid metaphases (1-3) were found in some cases in the negative (vehicle) control, positive control, untreated control or test item treated samples in the performed experiments. In one case endoreduplicated metaphase was also detected.

The observed spontaneous mutation frequency of this solvent was confirmed to be comparable with the general historical control range of other solvents, therefore it was considered to be acceptable.

The positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation demonstrating the sensitivity of the test system. The evaluated concentration range was considered to be adequate; at least three test item treated concentrations were evaluated in each assay. The tests were considered to be valid.

In conclusion, WARADUR LG did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and without metabolic activation. Therefore, WARADUR LG has not been shown to be clastogenic in this test system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 31 October 2017 to 1 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29. July 2016

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 liver extract from phenobarbital (PB) and β-naphthoflavone (BNF) induced male Wistar rats

Test concentrations with justification for top dose:

0, 0.41; 1.23; 3.70; 11.11; 33.33; 100; 300 μg/mL in all experiments
based on a preliminary cytotoxicity test with concentrations up to 2000 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: propylene glycol for test item, DMSO for positive controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension
- Cell density at seeding (if applicable): 2 x 10exp5 per mL

DURATION
- Exposure duration: 5 h (experiment 1 with and without metabolic activation, experiment 2 with metabolic activation), or 24 h (experiment 2 without metabolic activation)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days with 6-thioguanine
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days

SELECTION AGENT (mutation assays): 6-thioguanine

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates for each dose level

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

The test item was considered to be mutagenic in this assay, if the following criteria are met:
1. The assay is valid.
2. The mutant frequency at one or more doses is significantly greater than that of the relevant negative (vehicle) control (p<0.05).
3. Increase of the mutant frequency is reproducible.
4. There is a dose-response relationship.
Results which only partially met the criteria were dealt with on a case-by-case basis (historical control data of untreated control samples was taken into consideration, if necessary).
According to the relevant OECD guideline, the biological relevance of the results was considered first, statistical significance was not the only determination factor for a positive response

Statistics:

The heterogeneity of variance between groups was checked by Bartlett`s test. Where no significant heterogeneity was detected, a one-way analysis of
variance (ANOVA) was carried out. If the obtained result was significant, Duncan’s Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test. Data also were checked for a trend in mutation frequency with treatment dose using Microsoft Excel 2010 software.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: only slight cytotoxicity (minimum relative gowth: 84 %

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no large changes
- Effects of osmolality: no large changes
- Precipitation: see below

RANGE-FINDING/SCREENING STUDIES:
from 3.096 to 2000 μg/mL

Experiment 1 and 2: the following concentrations were examined: 300, 100, 33.33, 11.11, 3.70, 1.23 and 0.41 μg/mL.

In both experiments, insolubility (minimal precipitate; oily film) was detected at 300 – 0.41 μg/mL concentration range in the final treatment medium at the end of the treatment in the experiments with and without metabolic activation. Due to the characteristics of the test item (other recommended solvents were not appropriate for this test item) insolubility was observed in all examined concentrations, minimal amount of precipitate and oily film or in experiment 2 only minimal amount of oily film was detected at 0.41 μg/mL concentration, therefore this fact was considered to have no effect on the results. The precipitation did not interfere with the reading of the results.

Conclusions:

Under the conditions of this study, the test item was not mutagenic to mammalian cells in vitro.

Executive summary:

An in vitro mammalian cell assay was performed in CHO K1 Chinese hamster ovary cells at the hprt locus to evaluate the potential of WARADUR LG to cause gene mutation according to OECD testing guideline 476. Treatments were carried out for 5 hours with and without metabolic activation (±S9-mix) and for 24 hours without metabolic activation (-S9-mix). Propylene glycol was used as the vehicle (solvent) of the test item in this study.

Treatment concentrations for the mutation assays of the main tests were selected based on the results of a preliminary toxicity test as follows:

Assay 1

5-hour treatment in the presence of S9-mix: 300, 100, 33.33, 11.11, 3.70, 1.23 and 0.41 μg/mL

5-hour treatment in the absence of S9-mix: 300, 100, 33.33, 11.11, 3.70, 1.23 and 0.41 μg/mL

Assay 2

5-hour treatment in the presence of S9-mix: 300, 100, 33.33, 11.11, 3.70, 1.23 and 0.41 μg/mL

24-hour treatment in the absence of S9-mix: 300, 100, 33.33, 11.11, 3.70, 1.23 and 0.41 μg/mL

In the main assays, a measurement of the survival (colony-forming ability at the end of the treatment period) and viability (colony-forming ability at the end of the 7 day expression period following the treatment) and mutagenicity (colony forming ability at the end of the 7 day expression period following the treatment, in the presence of 6-thioguanine as a selective agent) was determined.

In Assays 1-2, insolubility was detected at 300 – 0.41 μg/mL concentration range in the final treatment medium at the end of the treatment in the experiments with and without metabolic activation. The precipitation did not interfere with the reading of the results. Minimal discoloured medium was observed with and without metabolic activation at several concentrations (with the exception of Assay 2 without metabolic activation). There were no large changes in pH and osmolality after treatment in any cases.

In Assay 1 and 2, in the presence of S9-mix (5-hour treatment), no marked cytotoxicity of the test item was observed. An evaluation was made using data of all seven concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data. No dose response to the treatment was observed (a trend analysis showed no effect of treatment).

In Assay 1, in the absence of S9-mix (5-hour treatment), no marked cytotoxicity of the test item was observed. An evaluation was made using data of all seven concentrations.

No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data.No dose response to the treatment was observed (a trend analysis showed no effect of treatment).

In Assay 2, in the absence of S9-mix (24-hour treatment), no marked cytotoxicity of the test item was observed, thus an evaluation was made using data of all seven concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data. No dose response to the treatment was observed (a trend analysis showed no effect of treatment).

Assay 2 confirmed the negative results seen in Assay 1 with and without metabolic activation.

The spontaneous mutation frequency of the negative (vehicle) control was confirmed to be comparable with the general historical control range of other solvents, therefore it was considered to be acceptable. The positive controls gave the anticipated increases in mutation frequency over the controls and were in good harmony with the historical data in all assays. Seven evaluated concentrations were presented in all assays. The cloning efficiencies for the negative controls at the beginning and end of the expression period were within the target range. The evaluated concentration ranges were considered to be adequate as they covered the range from insolubility to minimal insolubility. The overall study was considered to be valid.

In conclusion, no mutagenic effect of WARADUR LG was observed either in the presence or absence of a metabolic activation system under the conditions of this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Five bacterial mutagenicity test are available, which were performed according to OECD guideline 471 (Montan waxes, types WM 31, NaV 101, and LG; RL1) or equivalent or similar to that guideline (Montan waxes, types E and WE 4, RL1). None of them observed a mutagenic activity.

Genotoxic responses in vitro were not observed in reliable (guideline) tests on gene mutations (Montan waxes, types WM31, O, NaV101, S and LG, RL1) or chromosome mutations (Montan Waxes, types S, WM 31, O, NaV 101 and LG, RL1) in cultures of mammalian cells.

Due to the poor solubility of the test materials precipitation occurred in these tests at higher concentrations.

Based on these consistent negative results, no further testing is required.

Justification for classification or non-classification

Based on the findings of reliable studies with different types of Montan waxes, the Montan waxes category members have not to be classified for genotoxic effects according to Regulation (EC) No 1272/2008.