Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

FRESKOMENTHE did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

FRESKOMENTHE is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

FRESKOMENTHE is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 11-19, 2002
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
other: "Comission Directive 2000/32/EC, L1362000, Annexe 4D", dated May 19, 2000
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Name (as stated in the report): Freskomenthe
Batch: 9000466124
Expiration date: April 11, 2004
Purity: 99.3 %
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
liver microsomal activation
Test concentrations with justification for top dose:
Experiment I and II: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II without S9 mix: 10; 33; 100; 333; 1000; and 2500 µg/plate
Vehicle / solvent:
water deionised or DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylelene-diamine, 4-NOPD and 2-aminoanthracene, 2-AA
Details on test system and experimental conditions:
On the day of the experiment, the test item FRSKOMENTHE was dissolved in ethanol (purity > 99 %, MERCK, D-64293 Darmstad). The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
No precipitation of the test item occurred up to the highest investigated dose.

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 ena-bles substances to penetrate the cell wall more easily. A further mutation causes an inactivation of the 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 “urvB-minus”. In the strains TA 98 and TA 100 and TA 102 the R-factor plasmid pKM 101 carries the ampicillin resistance marker. The strain TA 102 does not contain the urvB--mutation and is excision repair proficient. Additionally, TA 102 contains the multicopy plasmid pAQ1 carrying the hisG428 mutation (ochre mutation in the hisG gene) and a tetracycline resistance gene.

Regular checking of the properties if the strains regarding the membrane permeability, ampicillin- and tetracycline-resistance as well as spontaneous mutation rates is performed in the laboratory of RCC Cytotest Cell Research according to Ames et al. (1). In this way it was ensured that the experimental conditions set down by Ames were fulfilled.

The bacterial strains TA 1535, TA 1537, and TA 100 were obtained from Ames (University of California, 94720 Berkeley, U.S.A.). The bacterial strain TA98 was obtained from E. Merck (D-64293 Darmstadt). The bacterial strain 102 was ob-tained from RCC Ltd. (CH-4332 Stein).

The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5% DMSO (MERCK, D-64293 Darm-stadt) in liquid nitrogen.
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 TA 102) 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.
Key result
Species / strain:
other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with FRESKOMENTHE at any concentration 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 historical range of positive controls was exceeded in strains TA 1535, Ta 100 without metabolic activation, and in strain TA 98 with metabolic activation in experiments I and II. These effects indicate the sensitivity of the strains rather than compromising the assay. In experiment II, with metabolic activation, the number of colonies did not quite reach the lower limit of our historical control data in strain TA 102 (solvent control). Since this deviation is rather small, this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase in 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:
Therefore, FRESKOMENTHE is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.
This study was performed to investigate the potential of FRESKOMENTHE to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. 
The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested on triplicate.
The test item was tested at the following concentrations:
 
Experiment I and II: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II without S9 mix: 10; 33; 100; 333; 1000; and 2500 µg/plate
 
Toxic effects were observed at higher concentrations with and without metabolic activation in nearly all strains used. 
Irregular background growth was observed with and without metabolic activation in nearly all strains used. 
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with FRESKOMENTHE 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.
Executive summary:

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.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
yes
Remarks:
see the "principle of method if other than guideline" field below
Principles of method if other than guideline:
In the dose range finding test cultures were fixed after 27 hours instead of 24 hours.
Evaluation: The purpose of the dose range finding test is to determine toxicity after a predetermined time period. A small deviation in the exposure time does not impact the study outcome and integrity.
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
Identification: FRESKOMENTHE
Appearance: Clear colourless liquid (determined by Charles River Den Bosch)
Batch: VE00411894
Test item storage: At room temperature protected from light
Stable under storage conditions until 11 February 2018 (expiry date)
Target gene:
The objective of this study was to evaluate FRESKOMENTHE for its ability to induce micronuclei in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix).
Species / strain / cell type:
lymphocytes: Human
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).
Test concentrations with justification for top dose:
Dose Range Finder:
At a concentration of 1600 μg/ml FRESKOMENTHE precipitated in the culture medium. In the dose range finding test blood cultures were treated with 17, 52, 164, 512, 1600 and 5000 μg FRESKOMENTHE/ml culture medium and exposed for 3 and 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix.
The pH and osmolarity of a concentration of 512 μg/ml were 7.69 and 454 mOsm/kg respectively (compared to 7.47 and 465 mOsm/kg in the solvent control).
Vehicle / solvent:
Ethanol
Untreated negative controls:
no
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:
Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in the international OECD guideline.
Blood was collected from healthy adult, non-smoking volunteers (aged 18 to 35 years). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined.
Rationale for test conditions:
Blood samples
Blood samples were collected by venepuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin (Vacuette, Greiner Bio-One, Alphen aan den Rijn, The Netherlands). Immediately after blood collection lymphocyte cultures were started.
Culture medium
Culture medium consisted of RPMI 1640 medium (Life Technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Life Technologies), L-glutamine (2 mM) (Life Technologies), penicillin/streptomycin (50 U/ml and 50 μg/ml respectively) (Life Technologies) and 30 U/ml heparin (Sigma, Zwijndrecht, The Netherlands).
Lymphocyte cultures
Whole blood (0.4 ml) treated with heparin was added to 5 ml or 4.8 ml culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin (Remel Europe Ltd., Dartford, United Kingdom) was added.
Evaluation criteria:
The following criteria for scoring of binucleated cells were used (1 - 2, 6):
- Main nuclei that were separate and of approximately equal size.
- Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
- Main nuclei that were linked by nucleoplasmic bridges.
The following cells were not scored:
- Trinucleated, quadranucleated, or multinucleated cells.
- Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).
The following criteria for scoring micronuclei were adapted from Fenech, 1996 (1):
- The diameter of micronuclei should be less than one-third of the main nucleus.
- Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
- Micronuclei should have similar staining as the main nucleus.
Statistics:
Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) and ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) were used for statistical analysis of the data.
A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with an Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, one-sided,
p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with an Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.
In case the Chi-square test shows that there are statistically significant differences between one or more of the test item groups and the vehicle control group a Cochran Armitage trend test
(p < 0.05) will be performed to test whether there is a significant trend in the induction.
Key result
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
The ability of FRESKOMENTHE to induce micronuclei in human peripheral lymphocytes was investigated in two independent experiments. The highest concentration analysed was selected based on toxicity, cytokinesis-block proliferation index of 55 ± 5%.
The cytokinesis-block proliferation indices of cultures treated with various FRESKOMENTHE concentrations or with the negative control items are presented in the report. The scores for the number of mono- and binucleated cells with micronuclei are presented in the report. Duplicate cultures are indicated by A and B. The individual data are described in the report. along with the statistical evaluations of the test results.
The number of mono- and binucleated cells with micronuclei found in the solvent control was within the 95% control limits of the distribution of the historical negative control database are presented in the report.
The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition colchicine also showed a statistically significant increase in the number of binucleated cells with micronuclei in the first cytogenetic assay. Furthermore, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
In the first cytogenetic assay, FRESKOMENTHE did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei.
In the second cytogenetic assay with a 24 hours continuous exposure time, FRESKOMENTHE induced a statistically significant increase in the number of mononucleated cells with micronuclei in the intermediate and high dose which was not dose dependent. Furthermore no trend was observed. Therefore this increase is not considered biologically relevant.
Remarks on result:
other: Negative
Conclusions:
In the first cytogenetic assay, FRESKOMENTHE did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei.
In the second cytogenetic assay with a 24 hours continuous exposure time, FRESKOMENTHE induced a statistically significant increase in the number of mononucleated cells with micronuclei in the intermediate and high dose which was not dose dependent. Furthermore no trend was observed. Therefore this increase is not considered biologically relevant.
Executive summary:

Finally, it is concluded that this test is valid and that FRESKOMENTHE is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 November 2016 to 06 February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH. S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use; CHMP/ICH/126642 ICH Consensus Guideline, Step 5 Guideline (Into Operation June 2012)
Principles of method if other than guideline:
The study integrity was not adversely affected by the deviations.
GLP compliance:
yes (incl. certificate)
Type of assay:
other: Mammalian Cell Gene Mutation Test with L5178Y Mouse Lymphoma Cells
Specific details on test material used for the study:
Identification FRESKOMENTHE
Appearance Clear colourless liquid (determined by Charles River Laboratories Den Bosch)
Batch: VE00426966
Stable under storage conditions until 19 February 2018 (expiry date)
Target gene:
The objective of this study was to evaluate the mutagenic potential of FRESKOMENTHE by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Test System L5178Y/TK+/--3.7.2C mouse lymphoma cells.
Rationale Recommended test system in international guidelines (e.g. OECD).
Source American Type Culture Collection, (ATCC, Manassas, USA) (2001).
Stock cultures of the cells were stored in liquid nitrogen (-196°C). The cultures were checked for mycoplasma contamination. Cell density was kept below 1 x 106 cells/ml.
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Based on the results of the dose range finding test, the following dose range was selected for the first mutagenicity test:
Without and with S9-mix: 12.5, 25, 50, 100, 150, 175, 200, 225, 250, 275 and 300 μg/ml exposure medium.
To obtain more information about the possible mutagenicity of FRESKOMENTHE, a second mutation experiment was performed in the absence of S9-mix with a 24-hour treatment period.
Based on the results of the dose range finding test, the following dose levels were selected for mutagenicity testing: 25, 50, 75, 100, 125, 140, 150, 160, 170, 180, 190 and 200 μg/ml exposure medium.
Vehicle / solvent:
The solvent for the test item was ethanol (Merck, Darmstadt, Germany).
Untreated negative controls:
yes
Remarks:
Solvent control
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
Per culture 8 x 106 cells (106 cells/ml for 3 hour treatment) or 6 x 106 cells (1.25 x 105 cells/ml for 24 hour treatment) were used. The cell cultures for the 3 hour treatment were placed in sterile 30 ml centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 rpm. The cell cultures for the 24 hour treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. Solvent and positive controls were included and the solvent control was tested in duplicate.In the first experiment, cell cultures were exposed for 3 hours to FRESKOMENTHE in exposure medium in the absence and presence of S9-mix. In the second experiment, cell cultures were exposed to FRESKOMENTHE in exposure medium for 24 hours in the absence of S9-mix.
For the 3 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 50 ml growth medium (R10-medium).
For the 24 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 20 ml growth medium (R10-medium). The cells in the final suspension were counted with the coulter particle counter.
For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 106 cells (where possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test item the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF).
For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
For determination of the mutation frequency (MF) a total number of 9.6 x 105 cells/concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 105 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.
Rationale for test conditions:
The study procedures described in this report are in compliance with the following guidelines:
Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Guideline no. 490: "Genetic Toxicology: In Vitro Mammalian Cell Gene Mutation Test Using the Thymidine Kinase Gene" (adopted 29 July 2016)
ICH. S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use; CHMP/ICH/126642 ICH Consensus Guideline, Step 5 Guideline (Into Operation June 2012)
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: Negative

First mutagenicity test

- Evaluation of toxicity :

In the absence of S9-mix, the dose levels of 225 to 300 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.

In the presence of S9-mix, the dose levels of 12.5 to 150 μg/ml showed no cytotoxicity and 175 to 225 μg/ml showed similar cytotoxicity. Therefore, the dose levels of 12.5 and 175 μg/ml were not regarded relevant for mutation frequency measurement. The dose level of 300 μg/ml was not used for mutation frequency measurement, since this dose level was too toxic for further testing. The dose levels selected to measure mutation frequencies at the TK-locus were: without S9-mix: 12.5, 25, 50, 100, 150, 175 and 200 μg/ml exposure medium and with S9-mix: 25, 50, 100, 150, 200, 225, 250 and 275 μg/ml exposure medium.

In the absence of S9-mix, the relative total growth of the highest test item concentration was 31% compared to the total growth of the solvent controls, see study plan

deviation 2. In the presence of S9-mix, the relative total growth of the highest test item concentration was 16% compared to the total growth of the solvent controls.

- Evaluation of the mutagenicity

No significant increase in the mutation frequency at the TK locus was observed after treatment with FRESKOMENTHE either in the absence or presence of S9-mix. The numbers of small and large colonies in the FRESKOMENTHE treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Second mutagenicity test

- Evaluation of toxicity

The dose levels of 140 to 160 μg/ml showed similar cytotoxicity. Therefore, the dose levels of 140 and 160 μg/ml were not regarded relevant for mutation frequency measurement.

The dose levels of 190 and 200 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. The dose levels selected to measure mutation frequencies at the TK-locus were: 25, 50, 75, 100, 125, 150, 170 and 180 μg/ml exposure medium. The relative total growth of the highest test item was 16% compared to the total growth of the solvent controls.

- Evaluation of mutagenicity

No significant increase in the mutation frequency at the TK locus was observed after treatment with the test item. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Conclusions:
The growth rate over the two-day expression period for cultures treated with ethanol was between 8 and 15 (3 hour treatment) and 71 and 75 (24 hour treatment).
In the absence of S9-mix, FRESKOMENTHE did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modification in the duration of treatment.
In the presence of S9-mix, FRESKOMENTHE did not induce a significant increase in the mutation frequency.
Executive summary:

Evaluation of the mutagenic activity of FRESKOMENTHE in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells.

This report describes the effects of FRESKOMENTHE on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in the absence of S9-mix with 3 and 24-hour treatment periods and in the presence of

S9-mix with a 3 hour treatment period (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

The study procedures described in this report were based on the most recent OECD and ICH guidelines.

Batch VE00426966 of FRESKOMENTHE was a clear colourless liquid. The test item was dissolved in ethanol.

In the first experiment, FRESKOMENTHE was tested up to concentrations of 200 and 275 μg/ml in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was 31 and 16% in the absence and presence of S9-mix, respectively. In the absence of S9-mix, above the dose level 200 μg/ml (225 μg/ml and above), the RSG (relative suspension growth) was below the acceptable limit of 10%. The test item did not precipitate in the culture medium at this dose level.

In the second experiment, FRESKOMENTHE was tested up to concentrations of 180 μg/ml in the absence of S9-mix. The incubation time was 24 hours. The RTG was 16%. The test item did not precipitate in the culture medium at this dose level. The mutation frequency found in the solvent control cultures was within the range of the acceptability criteria of this assay, except in the first experiment in which the mutation frequency of one of the solvent control cultures in the presence of S9-mix was not within the range of the acceptability criteria. Since the mutation frequency was just above the upper limit of the acceptability criteria range and clear negative results were obtained, this deviation in the mutation frequency had no effect on the validity of the results of the first mutation experiment.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, FRESKOMENTHE did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent

experiment with modification in the duration of treatment. In the presence of S9-mix, FRESKOMENTHE did not induce a significant increase in the mutation frequency.

It is concluded that FRESKOMENTHE is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Not required based on the complete in vitro standard package of assays.

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification