Reaction mass of N,N-ethanediylbis- Hexadecanamide, N-[2-[(1-oxohexadecyl)amino]ethyl]-Octadecanamide, Hexadecanoic acid, 12-hydroxystearic acid, reaction products with ethylenediamine, N,N-ethanediylbis-Octadecanamide and Octadecanoic acid, 12-hydroxystearic acid, reaction products with ethylenediamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Licolub FA 6 and its structural analogue, Licowax C, showed negative results in one study for the induction of gene mutations (bacterial reverse mutation assay) by frameshift or base-pair substitutions, one in vitro chromosome aberration study and one MLA with and without metabolic activation. The studies were performed according to recent OECD guidelines.

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:

2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and the Escherichia coli strain WP2 uvrA.

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 0.1; 0.3; 1; 3; 10; 33; 100; and 250 µg/plate.
Experiment II: 1; 3; 10; 33; 100; and 250 µg/plate
The maximum dose level was 250 µg/plate, limited by the solubility of the test item.

Vehicle / solvent:

On the day of the experiment, the test item was suspended in THF. The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
Precipitaion of the test item was observed in the overlay agar on the plates at 250 µg/plate in experiment II with and without S9 mix. The undissolved particles had no influence on the data recording.

Untreated negative controls:

yes

Remarks:

concurrent untreated

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-NOPD

Details on test system and experimental conditions:

CHARACTERISATION OF THE SALMONELLA TYPHIMURIUM STRAINS AND E. COLI STRAIN
The histidine dependent strains are derived from S. typhimurium strain LT2 through a mutation in the histidine locus. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98 and TA 100 the R-factor plasmid pKM 101 carries the ampicillin resistance marker.
Strain WP2 and its derivatives all carry the same defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent (Trp+) mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagen which substitute one base for another. Additionally, the uvrA derivative is deficient in the DNA repair process (excision repair damage). Such a repair-deficient strain may be more readily mutated by agents.

S9 (PREPARATION by RCC-CCR):
Phenobarbital/beta-Naphthoflavone induced rat liver S9 is used as the metabolic activation system. The S9 is prepared from 8 - 12 weeks old male Wistar Hanlbm rats, weight approx. 220 - 320 g induced by applications of 80 mg/kg b.w. Phenobarbital i. p. (Desitin; D-22335 Hamburg) and beta-Naphthoflavone p.o. (aldrich, D-89555 Steinheim) each on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCl solution (1+3) followed by centrifugation at 9000 g. Aliquotes of the supernatant are frozen and stored in ampoules at -80 °C. Small numbers of the ampoules can be kept at -20 °C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo(a)pyrene.
The protein concentration in the S9 preparation was 31.0 mg/mL (lot no. R 030306) in both experiments.

S9 MIX:
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15 % v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.

Rationale for test conditions:

concentrations were chosen based on results of a pre-experiment

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

No statistical evaluation of the data is required

Key result

Species / strain:

S. typhimurium TA 98

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

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

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

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

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:

valid

Positive 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:

valid

Positive controls validity:

valid

DISCUSSION OF RESULTS

The test item was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations limited by the solubility of the test item:

Pre-Experiment/Experiment I: 0.1; 0.3; 1; 3; 10; 33; 100; and 250 µg/plate.

Experiment II: 1; 3; 10; 33; 100; and 250 µg/plate

The plates incubated with the test item showed normal background growth up to 250 µg/plate with and without metabolic activation in both independent experiments.

No toxic effects evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed with and without metabolic activation in both experiments.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

The data in the negative control were slightly above our historical control range in strains TA 1535 (exp. I without S9 mix), TA 1537 (exp. II, without S9 mix), and WP2 uvrA (exp. I and II, with S9 mix). The data in the solvent control were slightly above our historical control range in strain TA 1535 (exp. I, without S9 mix). Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore the substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of the test substance to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations limited by the solubility of the test item:

Pre-Experiment/Experiment I: 0.1; 0.3; 1; 3; 10; 33; 100; and 250 µg/plate

Experiment II: 1; 3; 10; 33; 100; and 250 µg/plate

The plates incubated with the test item showed normal background growth up to 250 µg/plate with and without metabolic activation in both independent experiments.

No toxic effects evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed with and without metabolic activation in both experiments.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006

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:

other: in vitro chromosome aberration test

Target gene:

V79 cells (cell line from the lung of the Chinese Hamster)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

The highest concentration used in the cytogenetic experiments was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests requesting for the top concentration clear toxicity with reduced cell numbers or mitotic indices below 50 % of control, whichever is the lowest concentration, and/or the occurrence of precipitation. In case of non-toxicity the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest, if formulation in an appropriate solvent is possible.
With respect to the ability to formulate a homogeneous suspension of the test item, in the pre-test 164.2 µg/mL was applied as top concentration for treatment of the cultures. Test item concentrations between 1.3 and 164.2 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. Precipitation of the test item after 4 hrs treatment was observed at 20.5 µg/mL and above.
Using reduced cell numbers as an indicator for toxicity in the pre-test, no clear toxic effects were observed after 4 hrs treatment up to the highest applied concentration. Considering the toxicity data of the pre-test, 50 µg/mL (with and without S9 mix) was chosen as top concentration in Experiment I.
Dose selection of Experiment II was also influenced by test item toxicity and the occurrence of precipitation. In the range finding experiment clearly reduced cell numbers were observed after 24 hrs exposure with 81.8 µg/mL and above. Thereforek, 160 µg/mL was chosen as top treatment concentration for continuous exposure in the absence of S9 mix. In the presence of S9 mix 50 µg/mL was chosen as top treatment concentration with respect to the results obtained in Experiment I.
In Experiment II, in the absence of S9 mix, the cytogenetic evaluation of higher concentrations in the respective intervals was impossible due to strong test item induced toxic effects (reduced cell numbers and/or low metaphase numbers, partially paralleled by poor metaphase quality).

Vehicle / solvent:

Tetrahydrofurane (THF)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

TREATMENT

Exposure period 4 hours: The culture medium of exponentially growing cell cultures was replaced with serum-free medium (for treatment with S9 mix) or complete medium (for treatment without S9 mix) with 10 % FCS (v/v), containing the test item. For the treatment with metabolic activation 50 µL S9 mix per mL medium were used. Concurrent solvent and positive controls were performed. After 4 hrs the cultures were washed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time.
The "Saline G" solution was composed as follows (per litre):
NaCl 8000 mg
KCl 400 mg
Glucose x H2O 1100 mg
Na2HPO4 x 7 H2O 290 mg
KH2PO4 150 mg
pH was adjusted to 7.2

Exposure period 18 and 28 hours: The culture medium of exponentially growing cell cultures was replaced with complete medium (with 10 % FCS) containing different concentrations of the test item without S9 mix. The medium was not changed until preparation of the cells.
All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).

Preparation of the Cultures: Colcemid was added (0.2 µg/mL culture medium) to the cultures 15.5 hrs and 25.5 hrs, respectively after the start of the treatment. The cells on the slides were treated 2.5 hrs later, in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37 °C. After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3:1 parts, respectively). Per experiment two slides per group were prepared. After preparation the cells were stained with Giemsa.

Evaluation of Cell Numbers: For evaluation of cytotoxicity indicated by reduced cell numbers two additional cultures per test item and solvent control group, not treated with colcemid, were set up in parallel. These cultures were stained after 18 hrs and 28 hrs, respectively, in order to determine microscopically the cell number within 10 defined fields per coded slide. The cell number of the treatment groups is given in percentage compared to the respective solvent control.

Analysis of Metaphase Cells: Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 22 +/- 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells in 500 metaphase plates per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

Rationale for test conditions:

Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation.

Evaluation criteria:

Evaluation of Results:

A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
and/or
- no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Although the inclusion of the structural chromosome aberrations is the prupose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid:
A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of our historical control data (0.0 - 8.5 % polyploid cells).

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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

RESULTS AND DISCUSSION

The test item, suspended in tetrahydrofurane (THF), was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in the absence and the presence of metabolic activation by S9 mix.

Two independent experiments were performed. In Experiment I, the exposure period was 4 hrs with and without metabolic activation. In Experiment II the exposure period was 4 hrs with S9 mix and 18 hrs and 28 hrs without S9 mix. The chromosomes were prepared 18 hrs (Exp. I and II) and 28 hrs (Exp. II) after start of treatment with the test item.

In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.

In a range finding pre-test on toxicity cell numbers were scored 24 hrs after start of treatment as an indicator for cytotoxicity. Concentrations between 1.3 and 164.2 µg/mL were applied. No clear toxic effects were observed after 4 hrs treatment up to the highest applied concentration. In contrast, 24 hrs continuous treatment with 82.1 µg/mL and above in the absence of S9 mix induced strong toxic effects.

In the pre-experiment, precipitation of the test item in culture medium was observed after treatment with 20.5 µg/mL and above in the absence and presence of S9 mix. No relevant influence of the test item on the pH value or osmolarity was observed (solvent control 361 mOsm, pH 7.5 versus 368 mOsm and pH 7.4 at 164.2 µg/mL).

In the absence of S9 mix, precipitation of the test item in culture medium was observed, in Experiment I, after 4 hrs treatment with 25 µg/mL and above at preparation interval 18 hrs. In Experiment II, after 18 hrs continuous treatment, precipitation occurred with 20 µg/mL and above and after 28 hrs continuous treatment with 80 µg/mL and above.

In the presence of S9 mix, in Experiments I and II, precipitation of the test item in culture medium was observed after 4 hrs treatment with 12.5 µg/mL and above.

In this study, neither reduced mitotic indices nor reduced cell numbers of below 50 % of control could be observed up to the highest evaluated concentrations of the test item. However, in Experiment II, in the absence of S9 mix, concentrations showing clearly reduced mitotic indices were not evaluable for cytogenetic damage.

In both experiments, in the absence and presence of S9 mix, neither a statistically nor a biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.5 - 2.5 % aberrant cells, exclusive gaps) were close to the range of the solvent control values (0.5 - 1.5 % aberrant cells, exclusive gaps) and within the range of our historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps.

In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.9 - 3.8 %) as compared to the rates of the solvent controls (1.3 - 3.3 %).

In both experiments, EMS (300 or 400 µg/mL) or CPA (1.4 or 2.0 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) when tested up to precipitating concentrations and/or the highest evaluable concentration.

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.
Therefore the substance is considered to be non-clastogenic in this chromosome aberration test with and without S9 mix when tested up to precipitating concentrations and/or the highest evaluable concentration.

Executive summary:

The test item, suspended in tetrahydrofurane (THF), was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. The following study design was performed:

Without S9 mix				With S9 mix
	Experiment I	Experiment II		Experiment I	Experiment II
Exposure period	4 hrs	18 hrs	28 hrs	4 hrs	4 hrs
Recovery	14 hrs	-	-	14 hrs	24 hrs
Preparation interval	18 hrs	18 hrs	28 hrs	18 hrs	28 hrs

In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.

The highest applied concentration in the pre-test on toxicity (164.2 µg/mL) was chosen with regard to the ability to formulate a homogeneous suspension of the test item in an appropriate solvent.

Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation.

In Experiment I, in the absence and presence of S9 mix and in Experiment II, in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II, in the absence of S9 mix, concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Reference 3

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:

2010-03-10 to 2010-04-27

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

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase (TK) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin, sodium pyruvate and L-glutamin + 10% (v/v) heat-inactivated horse serum (24-hour exposure); for 3-hour exposure only 5% (v/v) heat-inactivated horse serum were included. Selective medium consisted of the basic medium + 20% (v/v) heat-inactivated horse serum and 5 µg/ml trifluorothymidine (TFT).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphtoflavone-induced rat liver S9

Test concentrations with justification for top dose:

Experiment 1:
In the absence and presence of 8% (v/v) S9-mix: 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8 and 1.0 µg/mL for 3h

Experiment 2:
In the absence of S9-mix: 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8 and 1.0 µg/mL for 24h
In the presence of 12% (v/v) S9-mix: 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8 and 1.0 µg/mL for 3h

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: hexane
- Justification for choice of solvent/vehicle: solubility/ability to suspend

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

hexane

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9-mix Migrated to IUCLID6: 7.5 µg/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

hexane

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9-mix Migrated to IUCLID6: 15 µg/mL and 5 µg/mL for 3h and 24h treatment, respectivley

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 and 24h, respectively
- Expression time (cells in growth medium): 2 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT) (Sigma)
- Selection time (if incubation with a selection agent): 11-12 days

NUMBER OF REPLICATIONS: 5 exposure plates

NUMBER OF CELLS EVALUATED: not applicable, number of mutants per well counted; 2000 cells/well inserted, total sum of mutants given

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth, relative survival, suspension growth, relative suspension growth, growth rate

Evaluation criteria:

A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls is between 65 and 120%. An acceptable number of surviving cells (10e6) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 x 10e-6 and ≤ 170 x 10e-6.
c) The growth rate (R) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32 and 180 (24 hours treatment).
d) The mutation frequency of MMS should not be below 500 x 10e-6, and for CP not below 700 x 10e-6.

The global evaluation factor (GEF) has been identified by the IWTG as the mean of the negative/solvent mutation frequeny (MF) distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than the MF of the controls + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if: a) None of the tested concentrations reaches a mutation frequency of the MF of the controls + 126. b) The results are confirmed in an independently repeated test.

Key result

Species / strain:

mouse lymphoma L5178Y cells

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
- Water solubility and precipitation: a slight precipitate of N,N’-EthyIenebis(stearamide)-commercial grade in the exposure medium was observed after 3 hours treatment at the concentration of 0.8 µg/mL and severe precipitate was observed at concentrations of 2.4 µg/mL and above. N,N'-Ethylenebis(stearamide)-commerciaI grade 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 Ending test was 24 µg/mL

RANGE-FINDING/SCREENING STUDIES:
Both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 24 µg/mL compared to the suspension growth of the solvent control.

COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the solvent treated control cultures were within the ranges of the historical controls.

Table 1: Cytotoxic and mutagenic responses

Treatment	Concentration [µg/mL]	Cloning efficiency [%]	Relative total growth [%]	Mutation frequency x 10^-6
				total	small colonies	large colonies
3 hours treatment without S9-mix
Solvent control (mean)	--	99	100	114	62	46
Test substance	0	116	126	110	59	43
	0.1	123	126	99	53	40
	0.2	113	111	110	63	41
	0.3	110	124	118	67	44
	0.4	113	139	128	60	59
	0.5	110	154	118	63	48
	0.6*	108	125	126	71	48
	0.8*	118	125	105	52	47
	1.0*	115	125	119	67	45
MMS	15	58	55	1227	675	354
3 hours treatment with 8% (v/v) S9-mix
Solvent control (mean)	--	106	100	102	55	42
Test substance	0	105	102	80	38	38
	0.1	94	89	119	66	46
	0.2	105	55	89	51	34
	0.3	93	76	113	64	43
	0.4	120	81	93	47	41
	0.5	121	127	97	55	37
	0.6*	101	86	87	53	30
	0.8*	97	94	72	44	25
	1.0*	121	112	79	45	30
CP	7.5	47	23	1431	876	351
24 hours treatment without S9-mix
Solvent control (mean)	--	93	100	58	29	27
Test substance	0	86	138	66	34	31
	0.1	72	108	77	43	32
	0.2	79	121	78	38	38
	0.3	98	150	47	26	19
	0.4	90	151	55	27	26
	0.5	81	133	69	37	30
	0.6*	80	125	67	35	31
	0.8*	90	143	61	37	22
	1.0*	88	131	63	34	27
MMS	5	66	81	629	357	211
3 hours treatment with 12% (v/v) S9-mix
Solvent control (mean)	--	106	100	82	50	29
Test substance	0	85	294	83	52	28
	0.1	107	58	109	48	55
	0.2	118	60	77	42	32
	0.3	91	161	88	50	34
	0.4	85	127	86	55	28
	0.5	101	146	74	49	23
	0.6*	86	42	103	66	32
	0.8*	93	155	67	40	25
	1.0*	62	66	130	55	70
CP	7.5	37	43	1905	1113	500

*precipitation of test substance in the exposure medium

The test substance did not induce a significant increase in mutation frequency in the absence or presence of metabolic activation in the first experiment. This result was confirmed in an independent experiment with a different concentration of the metabolic activation system or a longer exposure time without activation.

Conclusion:

The test substance did not induce gene mutation in mammalian cells in vitro under the conditions of the study.

Conclusions:

Based on results of a MLA performed according to OECD 476 the test item is considered to be not mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

not madatory

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Licolub FA 6 does not have to be not classified for mutagenicity since this substance and its strucutral analogue did not reveal any mutagenic effect in the bacterial reverse mutation assay, an in vitro chromosome aberration study and a MLA in the presence or absence of metabolic activation.