N6-(1-oxododecyl)-L-lysine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was tested in a study comparable to OECD Guideline 471 with Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA1538 as well as with Escherichia coli WP2uvrA using the preincubation method with and without metabolic activation. The test was performed with levels of 0 – 5000 ug/plate. A growth inhibition was observed at 5000 ug/plate in TA1535, TA98 and TA1537 without metabolic activation. A precipitation was not observed up to 5000 ug/plate both with and without metabolic activation. Therefore, the test item did not induce gene mutations in bacteria.

Test samples of Famex LL-T were assayed in an in vitro micronucleus test using human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals. Under the present test conditions, Famex LL-T tested up to the concentration of 2000 μg/mL medium in the absence and in the presence of metabolic activation employing two exposure times without S9 and one exposure time with S9 revealed no indications of chromosomal damage in the in vitro micronucleus test. The results for the vehicle controls were within historical control range. In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.

In a GLP study performed according to to OECD GL 490, the mutagenic potential of the test item Famex LL-T was evaluated based on quantification of forward mutations at the thymidine kinase locus of mouse lymphoma L5178Y/TK+/- cells. The microwell method of the “Mammalian Cell Gene Mutation Test” was used. Famex LL-T, both without and with S9 -mix, did not induce a relevant increase in the frequency of TFT-resistant mutants (MF) in the concentration range tested, and under the test conditions used. Thus, Famex LL-T did not show evidence of a significant, substance-specific induction of gene mutations in mouse lymphoma L5178Y/TK+/- cells, and is thus judged to be non-mutagenic in mammalian cells.

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:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Name: Nε-Lauroyl-L-Lysine
Supplier: Ajinomoto Co., Inc.
Molecular weight: 328.50
Lemon yellow powder with a purity of 95%

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

0, 5, 10, 50, 100, 500, 1000 and 5000 ug/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

furylfuramide

other: 2-aminoanthracene

Details on test system and experimental conditions:

The test was performed using the pre-incubation method with and without metabolic activation.

Evaluation criteria:

The results are judged as positive for inducibility of gene mutation when the number of revertant colonies per plate (mean) increases dose-dependently 2-fold or greater compared with that in the negative control. It is judged that the study has been performed satisfactorily when the number of revertant colonies in the negative and positive controls is within the range.

Statistics:

not further specified

Key result

Species / strain:

other: S. typhimurium TA98, TA100, TA1535, TA1537, TA1538 and Escherichia coli WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

The test item did not induce gene mutationn in bacteria.

Executive summary:

The test item was tested in a study comparable to OECD Guideline 471 with Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA1538 as well as with Escherichia coli WP2uvrA using the pre-incubation method with and without metabolic activation. The test was performed with levels of 0 – 5000 ug/plate. A growth inhibition was observed at 5000 ug/plate in TA1535, TA98 and TA1537 without metabolic activation. A precipitation was not observed up to 5000 ug/plate both with and without metabolic activation.

Therefore, the test item did not induce gene mutations in bacteria.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Designation: Famex LL-T
Batch no.: 130918
Characteristics: White to pale yellow powder
Storage conditions: Stored at +10°C to +25°C
Expiry date: September 17, 2017
Content: Main component: 99.9%; Accessory component: Water 0.1%

Species / strain / cell type:

mammalian cell line, other: human peripheral lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

250 - 2000 µg/mL
The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg Famex LL-T/mL medium were employed. No signs of cytotoxicity were noted up to the top concentration of 2000 µg/mL medium in the absence and presence of metabolic activation (24-hour or 4-hour exposure). Test item precipitation was noted macroscopically starting at a concentration of 31.6 µg Famex LL-T/mL medium in both experiments. No changes in pH or osmolality of the test item formulations compared to the negative control were noted up to the top concentration of 2000 µg/mL medium. Hence, 2000 µg/mL medium were employed as the top concentration for the genotoxicity tests without and with metabolic activation.

Vehicle / solvent:

Ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: colchicine

Details on test system and experimental conditions:

According to OECD GL

Evaluation criteria:

The assay demonstrates its ability to reliably and accurately detect substances of known aneugenic and clastogenic activity, with and without metabolic activation. Acceptance of a test is based on the following criteria:
The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database (Poisson-based 95% control limits). Where concurrent negative control data fall outside the 95% control limits, they may be acceptable for inclusion in the historical control data as long these data are not extreme outliers.
Concurrent positive controls induce responses that are compatible with those generated in the laboratory's historical positive control data base and produce a statistically significant increase compared with the concurrent negative control.
Adequate number of cells, cell proliferation criteria, and concentrations are analysable.
Vehicle control and untreated cultures give reproducibly low and consistent micronucleus frequencies. Data from vehicle and positive controls are used to establish historical control ranges. These values are used in deciding the adequacy of the concurrent vehicle controls or positive controls for an experiment.

Statistics:

Standard methods for statistics were applied.

Key result

Species / strain:

mammalian cell line, other: human peripheral lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Under the present test conditions, Famex LL-T tested up to the concentration of 2000 µg/mL medium in the absence and in the presence of metabolic activation employing two exposure times without S9 and one exposure time with S9 revealed no indications of chromosomal damage in the in vitro micronucleus test. The results for the vehicle controls were within historical control range. In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.

Executive summary:

Test samples of Famex LL-T were assayed in an in vitro micronucleus test using human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals. The test was carried out employing 2 exposure times without S9 mix: 4 and 24 hours, and 1 exposure time with S9 mix: 4 hours. The harvesting time was 20 hours after the end of exposure. The cytokinesis-block technique was applied.The test item was dispersed in ethanol to a stable suspension and diluted to the appropriate concentrations. The vehicle ethanol was employed as the negative control. The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg Famex LL-T/mL medium were employed. No signs of cytotoxicity were noted up to the top concentration of 2000 µg/mL medium in the absence and presence of metabolic activation (24-hour or 4-hour exposure). Test item precipitation was noted macroscopically starting at a concentration of 31.6 µg Famex LL-T/mL medium in both experiments. No changes in pH or osmolality of the test item formulations compared to the negative control were noted up to the top concentration of 2000 µg/mL medium. Hence, 2000 µg/mL medium were employed as the top concentration for the genotoxicity tests without and with metabolic activation.

In the main study, no signs of cytotoxicity were noted up to the top concentration of 2000 µg Famex LL-T/mL medium in the experiments without and with metabolic activation. Test item precipitation was noted macroscopically at all tested concentrations in all experiments. Mitomycin C (at 0.2 µg/mL) and colchicine (at 0.02 µg/mL) were employed as positive controls in the absence and cyclophosphamide (at 20 µg/mL) in the presence of metabolic activation.

Tests without metabolic activation (4- and 24-hour exposure)

The mean micronucleus frequencies of cultures treated with the concentrations of 250, 500, 1000 and 2000 µg Famex LL-T/mL medium in the absence of metabolic activation (4- and 24-hour exposure, respectively) ranged from 4.5 to 8.5 micronucleated cells per 1000 binucleated cells. There was no dose-related increase in micronuclei up to the top concentration of 2000 µg/mL medium. The frequency of micronucleated cells was within the historical control range of the untreated and vehicle controls. Vehicle controls should give reproducibly low and consistent micronucleus frequencies. In this test mean frequencies of 7.5 or 5.0 micronucleated cells per 1000 binucleated cells for the 4-hour and 24-hour exposure, respectively, were observed. The vehicle result was within the historical control ranges. In the positive control cultures, the mean micronucleus frequencies were increased to 20.5 or 22.5 micronucleated cells per 1000 binucleate cells for the 4-hour and 24-hour exposure, respectively. This demonstrated that Mitomycin C induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus.

Test with metabolic activation (4-hour exposure)

The mean micronucleus frequencies of cultures treated with the concentrations of 250, 500, 1000 and 2000 µg Famex LL-T/mL medium (4-h exposure) in the presence of metabolic activation ranged from 4.0 to 6.5 micronucleated cells per 1000 binucleated cells. There was no dose-related increase in micronuclei up to the top concentration of 2000 µg/mL medium. The frequency of micronucleated cells was within the historical control range of the untreated and vehicle controls. Vehicle controls should give reproducibly low and consistent micronucleus frequencies. In this test, a mean frequency of 4.5 micronucleated cells per 1000 binucleated cells was observed. The vehicle result was within the historical control ranges. In the positive control culture, the mean micronucleus frequency was increased to 19.0 micronucleated cells per 1000 binucleate cells for the 4-hour exposure. This demonstrated that cyclophosphamide induced significant chromosomal damage.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-10-18 to 2018-01-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

07-2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Name: Famex LL-T
Chemical name: N-Lauroyl-L-lysine
LOT No.: 130918
Appearance: White to pale yellow powder
Purity of test item: 99.9 %
Impurities: Water (0.1%)
Expiry date: 2018-09-17

Target gene:

Quantification of forward mutations at the thymidine kinase locus of mouse lymphoma L5178Y/TK+/- cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The study was conducted with L5178Y/TK+/- mouse lymphoma cells, clone 3.7.2C, which are heterozygous at the normally diploid TK locus. This permanent cell line was received from Boehringer Ingelheim Pharma GmbH & Co. KG on November 4, 2009. Freeze lots of the cells were tested for mycoplasma infection both by Boehringer Ingelheim Pharma GmbH & Co. KG (June 29, 2009) and Fraunhofer ITEM (November 27, 2009 and December 12, 2015) and were always found free of mycoplasma infection. The cells exhibit a diploid chromosome set of 48% 40, 42% 39, 8% 38 and 4% 37 chromosomes, as determined by methaphase spreads and analysis of 50 metaphases (September, 2015).
L5178Y/TK+/- cell stocks were cleansed before freezing to reduce number of spontaneously occurring TK+/- mutants. For that purpose the cells were cultured for 24 h in the presence of thymidine, hypoxanthine, methotrexate and glycine in order to poison TK+/- cells. Stocks of the cleansed cell line are stored at -80°C or in liquid nitrogen until use.
Cells were grown in plastic flasks at 37°C, 5 % CO2 and 90 % humidity. The standard growth medium consisted of RPMI-1640 medium with 25 mM HEPES, and 2 mM L-glutamine (ready to use culture medium) and 1 mM pyruvate, 10 % heat-inactivated HS, and penicillin/streptomycin (10000 U/10000 μg/ml, 1%) were added directly before use.

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

0, 0.9, 2.7, 8.2, 24.7, 74.1, 222.2, 666.7, and 2000 μg/ml for both 4 h, +/-S9-mix, and 24 h –S9-mix.
Single cultures per treatment and concentration.

Vehicle / solvent:

RPMI-1640 + 5% horse serum (R5; negative control)
Negative control with 250 μg/mL soy lecithin (S75) and 83 μg/ml Tween 80 (vehicle control)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

Determination of Cytotoxicity
For dose range finding, cytotoxicity of the test item Famex was determined with and without metabolic activation (S9-mix) in 4-h pre-tests using soy lecithin/Tween 80 as vehicle. The survival of the cells was estimated using eight concentrations of Famex up to the limit concentration of 2000 μg/ml. Aliquots of 5 x 106 cells of a proliferating cell culture were incubated for 4 h in 10 ml R5 with different concentrations of Famex in vehicle. Cells were incubated in the absence or presence of S9-mix in 25 cm2 cell culture flasks in the dark at 37°C with gently shaking, using an incubator. The different concentrations of the test item were generated by adding the prepared stock emulsions in a ratio of 1:80 to incubation medium, already containing the final cell number. Both in the absence and presence of S9-mix, the concentration range tested comprised 0 - 2000 μg/ml. At the end of the incubation period, the cells were washed twice, re-suspended in growth medium with 10% heat-inactivated HS, counted, and then serially diluted to 8 cells/ml with growth medium supplemented with 20% heat-inactivated HS. Two hundred μl of this cell suspension were subsequently plated in each well of one 96-well plate per concentration. Cells were then cultured for further 6 days. After that, visible colonies were counted and plating efficiency (PE) and relative survival (RS) were calculated. In addition, relative cell counts post-exposure were documented.
Concentrations of the Test Item in the Main Tests
The test item Famex exhibits limited water solubility and had thus to be tested as cloudy, but homogenous emulsions. Concentrations of Famex for the main test were based on the pre-tests on cytotoxicity. In the case of cytotoxicity, the test concentrations should cover a range from the maximum (see above) to little or no cytotoxicity, and the maximum concentration should result in about 10-20 % (but not less than 10 %) relative total growth (RTG), as compared to the vehicle control. However, Famex LL-T did not show marked cytotoxicity in the pre-tests on cytotoxicity, and it could be estimated that even at the limit concentration of 2000 μg/ml, 10-20 % RTG will not be reached. Additionally, no marked changes in pH and osmolality were seen. Therefore, it was decided to use the broad panel of concentrations used in the pre-tests (0 - 2000 μg/ml) also for the main experiments, and to use single cultures to be able to handle such a broad range of concentrations. All main tests comprised all eight test item concentrations and the respective reference items.
Test Procedure
Five days before starting the experiments, an exponentially growing stock culture of cleansed cells in growth medium was set up so as to provide an excess of cells on the morning of the experiment. Cells were splitted twice before treatment.
Preparation of the test item: The 80-fold concentrated vehicle stock emulsion and the Famex stock emulsions were prepared. The different treatment concentrations of the test item were generated thereafter by addition of 250 μl of the stock emulsions to 19.75 ml of pre-warmed incubation medium, already containing the final cell number, to get a final incubation volume of 20 ml. The positive reference items were directly added to the cell suspensions in incubation medium.
Treatment of target cells: Aliquots of 10 x 106 cells of a proliferating culture were incubated for 4 or 24 h at 37 ± 2°C in 20 ml incubation medium (R5) with different concentrations of the Famex vehicle emulsion, the positive reference items CP or MMS, or the used vehicle. Treatment was carried out with (4h of incubation) or without S9-mix (4 and 24 h of incubation) in sterile 25 cm2 cell culture flasks, with gently shaking in the dark.
Expression of the mutant phenotype: At the end of the exposure periods, cells were washed with R5, collected by centrifugation, and re-suspended in growth medium with 10% heat-inactivated HS (R10). After counting, two dilutions were prepared: 8 cells/ml growth medium with 20% heat-inactivated HS (R20) for determination of direct post-treatment cytotoxicity/viability and 2 x 105 cells/ml in standard growth medium (R10) for the expression of the mutant phenotype. For expression of the mutant phenotype, cells were cultured for additional two days in culture flasks, starting with 6 x 106 cells in 30 ml of R10. Cells were counted and population was again adjusted to 6 x106 cells after 24 h. To estimate post-treatment cytotoxicity (survivor I), cells were plated after treatment in R20 at a density of approximately 1.6 cells/well using two 96-well plates per treatment group. Cells were grown for additional 5-7 days until colonies were detectable and plating efficiency (PE) and relative survival (RS) were subsequently calculated.
Selection of the mutant phenotype: At the end of the expression period, TK-/- mutants, exhibiting a 5-trifluorothymidine (TFT)-resistant phenotype were detected by plating cells at a density of about 2 x 103 cells per well on four 96-well plates per treatment group. Cells were grown for 10-13 days in selective medium, consisting of growth medium with 20% heat-inactivated HS and 3 μg/ml of TFT. After the expression period, viability (survivor II) was also determined by plating cells at a density of about 1.6 cells/well on two 96-well plates per treatment group in non-selective growth medium (growth medium with 20% heat-inactivated HS, but without TFT). Cells were grown until colonies were detectable, and the plating efficiency (PE viable) and relative survival (RS) were calculated. PE viable data were also used to determine mutant frequencies (MF).
Evaluation: Quantification of the viable colonies of survivor I and II was performed microscopically 5-7 days after plating. TFT-resistant colonies were evaluated microscopically 10-13 days after plating. MF was then calculated. Viability was also judged by cell counting immediately after treatment and over the 2-day expression period. Suspension growth (SG) and relative total growth (RTG) were subsequently calculated. By referring to the respective OECD guideline, colony sizing has to be performed on at least one of the test item concentrations (the highest positive concentration) and the negative/vehicle and positive controls, using the criteria of small and large colonies, if the test item is positive/mutagenic. If the test item is negative, colony sizing has only to be done on negative and positive controls. Nevertheless, in this study, colony sizing was performed for all treatment groups. Colonies were counted as "large colonies" if they covered more than 1/4 of the well surface and exhibited a broad-faced growth. The thickness of large colonies is generally not more than 1 or 2 cells. Colonies were counted as "small colonies" if they covered less than 1/4 of the well surface, exhibited a compact growth, and had a thickness of more than 2 cells.
Plate Scoring
Quantification of the viable colonies of survivor I and II was performed microscopically at 40-fold magnification, 5-7 days after plating. The number of positive and negative wells and the total number of countable wells (192 per treatment group and experiment, 384 in total) were recorded. TFT-resistant colonies were evaluated microscopically at 40-fold magnification 10-13 days after plating (the 24 h experiment was evaluated after 13 days, because of unplanned absence of the respective technician). The number of positive and negative wells was recorded, together with the total number of countable wells (384 per treatment group and experiment, 768 in total). Colonies seen in the TFT-selection plates were qualified as large and small colonies for all treatment options, and the number of large and small colonies was also recorded.

Evaluation criteria:

see below

Statistics:

Standard statistical methods were used

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

Both the test item and the used vehicle did not lead to relevant changes in pH and osmolality, compared to R5 medium (negative control).

After 4 h without S9-mix, the test item did not induce relevant reduction in suspension growth (SG) and relative total growth (RTG), and cell growth was thus not markedly impaired up to the limit concentration of 2000 μg/ml, when compared to the vehicle control. In the presence of S9-mix, the test item slightly reduced, in a scattered way, SG with a maximum reduction from 15.99 for the vehicle control to 12.45 for 74.1 μg/mL of the test item. But, RTG demonstrated no relevant cytotoxicity.

In the absence of S9-mix, the vehicle itself showed some growth inhibitory potential, compared to the negative control. This was even more obvious after 24 h of incubation, and finally led to repetition of the respective main test I, because the lower acceptability limit for SG for vehicle controls (32) was not reached. In the repetition experiment (main test II), a slightly too low SG value for the vehicle control was again noted, amounting to 29.66. This seemed to represent an acute effect, as relative survival (RS; survivor II) was only slightly reduced, compared to the negative control. There was no possibility to use another vehicle, as known standard vehicles did not enable generation of stable and homogenous test item emulsions, as the test item exhibits no water solubility.

After 24 h, the test item mediated slight, not concentration-dependent, reduction in SG to about 80% of the vehicle control. For 666.7 and 2000 μg/mL, however, no decrease in cell growth was obvious, and RTG remained almost unchanged for all concentrations tested.

The vehicle controls exhibited acceptable mean mutant frequencies (MF) for all treatment regimes, which were all within the normal range (50-170 mutants/10E+6 viable cells) for L5178Y/TK+/- cells at the TK+/- locus. The positive controls MMS (-S9-mix) and CP (+S9-mix) induced marked and relevant increases in MF (increase in small colony MF of at least 150*10E-6 above the concurrent negative controls), indicating satisfactory performance of the test and sufficient activity of the metabolising system. Based on the concept of the “Global Evaluation Factor” (Moore et al. (2006) Mouse lymphoma thymidine kinase gene mutation assay: follow-up meeting of the International Workshop on Genotoxicity Testing - Aberdeen, Scotland, 2003 - Assay acceptance criteria, positive controls, and data evaluation. Environmental and Molecular Mutagenesis 47(1), 1-5), the test item did not induce relevant increases in MF at the TK+/- locus of L5178Y/TK+/- cells both after 4 (+/- S9-mix) and 24 h (- S9-mix). All MFs fell within the range proposed by Moore et al. (2006) for negative/vehicle controls (50 - 170 mutants/10E+6 viable cells), and MF for the 4 h experiments did not exceed the historical range for negative/vehicle controls (4 h -S9-mix: 54.0 to 169.7; 4 h +S9-mix: 49.7 to 144.2) of Fraunhofer ITEM. In the 24 h main test II, only for 222.2 and 2000 μg/mL test item, MF values were noted, which slightly exceeded the historical range (24 h –S9-mix: 69.2 to 152.5), amounting to 164.4 and 156.9*10E-6, respectively, but did not represent relevant increases, as MF for the concurrent vehicle control amounted to 148.8.

Conclusions:

The test item Famex LL-T, both without and with S9-mix, did not induce a relevant increase in the frequency of TFT-resistant mutants (MF) in the concentration range tested, and under the test conditions used. Thus, Famex LL-T did not show evidence of a significant, substance-specific induction of gene mutations in mouse lymphoma L5178Y/TK+/- cells, and is thus judged to be non-mutagenic in mammalian cells.

Executive summary:

This GLP study was conducted in compliance with the OECD GL 490 and this in vitro test was performed to evaluate the mutagenic potential of the test item Famex LL-T based on quantification of forward mutations at the thymidine kinase locus of mouse lymphoma L5178Y/TK+/- cells. The microwell method of the “Mammalian Cell Gene Mutation Test” was used.

Famex LL-T, both without and with S9 -mix, did not induce a relevant increase in the frequency of TFT-resistant mutants (MF) in the concentration range tested, and under the test conditions used. Thus, Famex LL-T did not show evidence of a significant, substance-specific induction of gene mutations in mouse lymphoma L5178Y/TK+/- cells, and is thus judged to be non-mutagenic in mammalian cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Concerning a possible mutagenic/genotoxic potential, Famex LL-T was tested negative in three different in vitro studies. Therefore, there is no need for classification and labelling of the test item according to CLP Regulation 1272/2008/EG.