Reaction mass of 2,6-Bis[(dimethylamino)methyl]-4-(1-{3-[(dimethylamino)methyl]-4-hydroxyphenyl}-1-methylethyl)phenol and 4-(1-{3,5-Bis[(dimethylamino)methyl]-4-hydroxyphenyl}-1-methylethyl)-2,6-bis[(dimethylamino)methyl]phenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

HPP 12879-1 shows neither mutagenic effects in a Salmonella/microsome test (strains TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2; +/- S9 mix; Sokolowski, 2015a) or in a HPRT test (V79 cells; +/- S9 mix; Wollny, 2016) nor clastogenic effects in a micronucleus test in vitro (human lymphocytes; +/- S9 mix; Sokolowski, 2015b).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

March to April 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Stable in ethanol (0.01-423 mg/ml) for 24hrs at room temperature (Currenta File No.: 2014/0048/08; date: 2015-03-24)


FORM AS APPLIED IN THE TEST (if different from that of starting material): On the day of the experiment, the test item was dissolved in ethanol. All formulations were prepared freshly before treatment and used within two hours of preparation.

Target gene:

mutant histidine gene

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

plate incorporation assay (experiment I):
3, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate with and without S9 mix
preincubation assay (experiment II):
33, 100, 333, 1000, 2500, 5000 µg/plate with and without S9 mix

The test was performed up to and including the limit dose of 5000 µg/plate. In both experiments no precipitation of the test item in the overlay agar in the test tubes occurred up to and including the highest investigated dose. The test item precipitated in the overlay agar on the incubated agar plates in experiment I at 5000 μg/plate in all strains without S9 mix. The undissolved particles had no influence on the data recording.
Toxic effects, evident as reduced background growth, were described for all test strains in experiment II without and with S9 mix at 5000 µg/plate.
Toxic effects, evident as a reduction in the number of revertants below the induction factor of 0.5) were observed in experiment I for TA 100 without S9 mix at 5000 µg/plate. In experiment II at 5000 µg/plate without S9 mix in TA 1535, TA 98 and TA 100 and with S9 mix in TA 100.

Vehicle / solvent:

Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.

DETERMINATION OF TOXICITY
- Method: Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

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:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: yes/no, but tested up to the limit concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: yes/no, but tested up to the limit concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: yes/no, but tested up to the limit concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: yes/no, but tested up to the limit concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: yes/no, but tested up to the limit concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

negative

Executive summary:

The test item was evaluated in an Ames Test on Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA, performed according to OECD TG 471. In both experiments no precipitation of the test item in the overlay agar in the test tubes occurred up to and including the highest investigated dose. The test item precipitated in the overlay agar on the incubated agar plates in experiment I at 5000 μg/plate in all strains without S9 mix. The undissolved particles had no influence on the data recording. The plates incubated with the test item at 5000 µg/plate showed reduced background growth at 5000 μg/plate in experiment II (+/- S9 mix). Toxic effects, evident as a reduction in the number of revertants, occurred in some test groups with and without metabolic activation at 5000 µg/plate in both experiments. The test material was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test. Positive controls increased the mutant counts to well over those of the negative controls, thus demonstrated the system´s sensitivity.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

June to July 2015

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:

2014

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Stable in ethanol (0.01-423 mg/ml) for 24hrs at room temperature (Currenta File No.: 2014/0048/08; date: 2015-03-24)


FORM AS APPLIED IN THE TEST (if different from that of starting material): Stock formulations of the test item and serial dilutions were made in ethanol. The final concentration of ethanol in the culture medium was 0.5 %. All formulations were prepared freshly before treatment and used within two hours of preparation.

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbecco's Modified Eagle Medium/Ham's F12 (mixture 1:1) supplemented with 200 mM GlutaMAX TM, penicillin/streptomycin 100 U/mL/100 µg/mL, PHA 3 µg/mL, 10 % fetal bovine serum, 10 mM HEPES, and heparin 125 U.S.P.-U/mL.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from the liver of Phenobarbital/beta-naphthoflavone induced rats.

Test concentrations with justification for top dose:

Exp. I, (4 hrs): µg/mL (without and with S9 mix): 13.0, 22.7, 39.8, 69.6, 121.9, 213.2, 373.2, 653.1, 1142.9 and 2000 µg/mL
Exp. II, (20 hrs): 13.0, 22.7, 39.8, 69.6, 121.9, 213.2, 373.2, 653.1, 1142.9 and 2000 µg/mL (solely without S9 mix) and (4hrs): 121.9, 213.2, 373.2, 653.1, 1142.9 and 2000 µg/mL (solely with S9 mix)

The following concentrations were selected for reading:
Exp. I (without and with S9 mix): 373.2, 653.1 and 1142.9 µg/mL
Exp. II: (20 hrs, without S9 mix): 121.9, 213.2, 373.2 mg/mL; (4hrs, with S9 mix): 373.2, 653.1 and 1142.9 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcin

Details on test system and experimental conditions:

Blood samples were drawn from healthy non-smoking donors not receiving medication. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with phytohemeagglutinine (PHA) and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes. The lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours.

Pulse exposure
About 48 hrs after seeding 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 μL S9 mix per mL culture medium was added. After 4 hrs the cells were gently centrifugated, separated from the supernatant, washed and resuspended in complete culture medium for a 16-hour recovery period. After this period Cytochalasin B (4 μg/mL) was added and the cells were cultured another approximately 20 hours until preparation.

Continuous exposure (without S9 mix)
About 48 hrs after seeding 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with complete medium (with 10 % FBS) containing the test item. After 20 hours the cells were gently centrifugated, separated from the supernatant, washed and resuspended in complete culture medium. Cytochalasin B (4 μg/mL) was added and the cells were cultured another approximately 20 hours until preparation.

STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED: At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is characterized by the percentages of reduction in the CBPI (Cytokinesis-block proliferation index) in comparison with the controls (% cytostasis) by counting 500 cells per culture in duplicate.

DOSE SELECTION
Dose selection was performed according to the current OECD Guideline for the in vitro micronucleus test. The highest test item concentration should be 10 mM, 2 mg/mL, or 2 μL/mL, whichever is the lowest. At least three test item concentrations should be evaluated for cytogenetic damage.
2000.0 μg/mL were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 13.0 to 2000.0 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, precipitation of the test item was observed at the end of treatment at 1142.9 μg/mL and above in the absence and presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
Using a reduced Cytokinesis-block proliferation index (CBPI) as an indicator for toxicity, no cytotoxic effects were observed in Experiment I after 4 hours treatment in the absence and presence of S9 mix. Therefore, 2000.0 μg/mL were chosen as top treatment concentration for Experiment II. The cytogenetic evaluation of concentrations in Experiment II (20 hrs, without S9 mix) higher than 373.2 µg/mL was impossible due to strong test item-induced toxic effects (low cell numbers).

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in all of the experimental conditions examined:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data
The test item is then considered unable to induce chromosome breaks and/or gain or loss in this test system.
Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data
When all of the criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.

Statistics:

Statistical significance will be confirmed by using the Chi-squared test (α < 0.05) using the validated R Script CHI2.Rnw for those values that indicate an increase in the number of cells with micronuclei compared to the concurrent solvent control. Other statistical methods may be used if appropriate.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis.
The highest treatment concentration in this study, 2000.0 μg/mL was chosen with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.
In Experiment I and II precipitation of the test item in the culture medium was observed at 1142.9 μg/mL and above in the absence and presence of S9 mix at the end of treatment.
No relevant influence on osmolarity was observed. The pH was adjusted to physiological values using small amounts of 2 M HCl.
In both cytogenetic experiments, in the absence and presence of S9 mix, no cytotoxicity indicated as cytostasis was observed up to and including the highest applied concentration.
In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of micronucleated cells was observed after treatment with the test item.
In both experiments, either Demecolcin (125.0 ng/mL), MMC (1.0 μg/mL) or CPA (15.0 μg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

Conclusions:

negative

Executive summary:

The test item dissolved in ethanol, was assessed for its potential to induce micronuclei in human lymphocytes in vitro according to OECD TG 487. No cytotoxicity indicated as cytostasis was observed up to the limit concentration in the absence and presence of S9 mix. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of micronucleated cells was observed after treatment with the test item. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei. Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

September to October 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1997)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Stable in ethanol (0.01-423 mg/ml) for 24hrs at room temperature (Currenta File No.: 2014/0048/08; date: 2015-03-24)


FORM AS APPLIED IN THE TEST (if different from that of starting material): On the day of the experiment immediately before treatment), the test item was dissolved in ethanol.

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM containing Hank's salts supplemented with 10 % FBS (except during 4 hour treatment), neomycin (5 µg/mL) and amphotericin B (1 %); for the selection of mutant cells the complete medium was supplemented with 11 µg/mL 6-thioguanine.
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from the liver of phenobarbital/beta-naphthoflavone induced rats.

Test concentrations with justification for top dose:

Exp. I: 4 hours exposure, without and with S9-mix , 250.0, 500.0, 1000.0, 2000.0, 3000.0 and 4000.0 µg/mL
Exp. II: 24 hours exposure, without S9-mix: 31.3, 62.5, 125.0, 250.0, 500.0 and 750.0 µg/mL; 4 hours exposure, with S9-mix: 250.0, 500.0, 1000.0, 2000.0, 3000.0 and 4000.0 µg/mL

The following concentrations were selected for reading:
Exp. I with and wihout S9-mix: 500.0, 1000.0, 2000.0, 3000.0 and 4000.0 µg/mL
Exp. II without S9 mix: 31.3, 62.5, 125.0 and 250.0 µg/mL.
Exp. II with S9 mix: 500.0, 1000.0, 2000.0, 3000.0 and 4000.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol (The final concentration of ethanol in the culture medium was 0.5% (v/v)).
- Justification for choice of solvent/vehicle: The solvent was chosen based on its compatibility with the test item, its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE). The highest concentration used in the pre-test (4000 μg/mL) was limited by the solubility properties of the test item in ethanol and aqueous medium. Test item concentrations between 31.3 μg/mL and 4000 μg/mL were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. Strong toxic effects were noted after 24 hours treatment at 500.0 μg/mL and above without metabolic activation. After 4 hours treatment no cytotoxicity was determined up to the maximum concentration with and without metabolic activation.The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Precipitation occurred at 4000 μg/mL after 4 hours treatment with and without metabolic activation.There was no relevant shift of osmolarity of the medium even at the maximum concentration of the test item. In the pre-experiment the pH value at concentrations of 125.0 mg/mL and above with and without metabolic activation was neutralised with 2M hydrochloric acid. In experiment I (with and without metabolic activation) the pH was neutralised with 2M sodium hydroxide at concentrations of 1000.0 μg/mL and above. In experiment II the pH was neutralised at 500.0 μg/mL and above with metabolic activation. There was no relevant shift of osmolarity of the medium at the maximum concentration of the test item in the pre-experiment.The dose range of experiment I was set according to cytotoxicity noted in the pre-experiment. The concentration range of the second experiment was chosen based on the results of the pre-experiment (without metabolic activation) and the first main experiment (with metabolic activation). The individual concentrations were generally spaced by a factor of 2.0. Closer spacing was used at high concentrations to cover the toxic range more closely.To overcome problems with possible deviations in toxicity the main experiments were started with more than four concentrations.
The cultures at the lowest concentration of experiment I with and without metabolic activation and experiment II with metabolic activation were not continued as a minimum of only four analysable concentrations is required by the guidelines. The cultures at the highest two concentrations of experiment II without metabolic activation were not continued due to exceedingly severe cytotoxic effects.

The main test was performed in two independent experiments. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

Culture Medium:
For seeding and treatment of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, 10% FBS (except during 4 hour treatment), neomycin (5 μg/mL) and amphotericin B (1%). For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2.
Seeding:
Two to three days after sub-cultivation stock cultures were trypsinized at 37 °C for 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10% FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2% in PBS. Prior to the trypsin treatment the cells were rinsed with PBS buffer containing 200 mg/L EDTA (ethylene diamine tetraacetic acid). Approximately 1.5×106 (single culture) and 5×102 cells (in duplicate) were seeded in plastic culture flasks. The cells were grown for 24 hours prior to treatment.

Treatment:
After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 μL/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. After 4 hours this medium was replaced with complete medium following two washing steps with "saline G". In the second experiment the cells were exposed to the test item for 24 hours in complete medium, supplemented with 10% FBS, in the absence of metabolic activation. The pH was adjusted to 7.2. The colonies used to determine the cloning efficiency (survival) were fixed and stained approx. 7 days after treatment as described below. Three or four days after treatment 1.5×106 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5×105 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2 for about 8 days. The colonies were stained with 10% methylene blue in 0.01% KOH solution. The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No relevant cytotoxic effects indicated by a cloning efficiency I or relative cell density below 50% occurred in both main experiments up to the maximum concentration with and without metabolic activation.
No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. The mutation frequency remained well within the historical range of solvent controls. The threshold of three times the corresponding solvent control was not reached or exceeded.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in culture I of the first experiment with metabolic activation, and in culture I of the second experiment with metabolic activation. As the mutation frequency did not exceed the threshold indicated above, the statistical results were judged as biologically irrelevant.
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 7.9 up to 23.2 mutants per 106 cells; the range of the groups treated with the test item was from 7.1 up to 43.8 mutants per 106 cells.
EMS (150 μg/mL) and DMBA (2.2 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies

Remarks on result:

other: 4 hours treatment

Conclusions:

negative

Executive summary:

A study according to OECD TG 476 was conducted in order to investigate the potential of the test item to induce gene mutations at the HPRT locus in mammalian cells. For that purpose, V79 cells were exposed to the test item in the first experiment for 4 hours with and without metabolic activation and in the second experiment for 4 hours with and for 24 hours without metabolic activation. The highest applied concentration (4000 μg/mL) was limited by the solubility properties of the test item in ethanol and aqueous medium. The concentration range of the second experiment (31.3 - 250.0 µg/mL) without metabolic activation (24 h treatment) was limited by cytotoxicity. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations in mammalian cells.  

 

 

 

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The test item was evaluated in an Ames test on Salmonella typhimurium strainsTA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA, performed according to OECD TG 471 (Sokolowski, 2015a). In both experiments no precipitation of the test item in the overlay agar in the test tubes occurred up to and including the highest investigated dose. The test item precipitated in the overlay agar on the incubated agar plates in experiment I at 5000 μg/plate in all strains without S9 mix. The undissolved particles had no influence on the data recording. The plates incubated with the test item at 5000 µg/plate showed reduced background growth at 5000 μg/plate in experiment II (+/- S9 mix). Toxic effects, evident as a reduction in the number of revertants, occurred in some test groups with and without metabolic activation at 5000 µg/plate in both experiments.The test material was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test. Positive controls increased the mutant counts to well over those of the negative controls, thus demonstrated the system´s sensitivity.

The test item dissolved in ethanol, was assessed for its potential to induce micronuclei in human lymphocytes in vitro according to OECD TG 487 (Sokolowski, 2015b). No cytotoxicity indicated as cytostasis was observed up to the limit concentration in the absence and presence of S9 mix. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of micronucleated cells was observed after treatment with the test item. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei. Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.

A study according to OECD TG 476 was conducted in order to investigate the potential of the test item to induce gene mutations at the HPRT locus in mammalian cells (Wollny, 2016). For that purpose, V79 cells were exposed to the test item in the first experiment for 4 hours with and without metabolic activation and in the second experiment for 4 hours with and for 24 hours without metabolic activation. The highest applied concentration (4000 μg/mL) was limited by the solubility properties of the test item in ethanol and aqueous medium. The concentration range of the second experiment (31.3 - 250.0 µg/mL) without metabolic activation (24 h treatment) was limited by cytotoxicity. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations in mammalian cells.

Justification for classification or non-classification

Based on the available study results (negative in Ames test, HPRT test and micronucleus test in vitro) a classification according to Regulation (EC) No. 1272/2008 (CLP) is not required.