Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Key, gene mutation in bacteria, OECD 471, GLP, with and without S9: negative with and without S9

Key, Chromosomal abberation, OECD TG 473, GLP, with and without S9: negative with and without S9

Key, mammalian cell gene mutation, OECD 476, GLP, with and without S9: negative with and without S9

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 JAN 1997 - 28 FEB 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was performed in compliance with the Good Laboratory Practice (GLP) regulations (revised in 1997, ENV/MC/CHEM(98)17).The method followed that described in the OECD Guidelines for Testing of Chemicals (Adopted: 4 April 1984) No 471 "Bacterial Reverse Mutation Test".
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1995
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
HIS operon (S. thyphimurium), TRY operon (E. coli)
Species / strain / cell type:
S. typhimurium TA 1535
Remarks:
his G 46, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 1537
Remarks:
his C 3076, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 98
Remarks:
his D 3052, uvrB, rfa + R-factor
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 100
Remarks:
his G 46, uvrB, rfa + R-factor
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 102
Remarks:
his G 428, rfa + R-factor
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
E. coli WP2
Remarks:
his C 3076, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the tryptophan operon
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
rat liver homogenate (S9 mix) with standard co-factors with metabolic activation (Aroclor)
- source of S9 : The batches of the liver homogenate fraction (S9) used in this study were prepared at the Institute of Toxicology (room U9/1092) of MERCK KGaA, Darmstadt, from October 28 to November 01, 1996. The acceptance criterium was entirely met.
- method of preparation of S9 mix : Thom (Wistar) rats (aged 6-8 weeks) were given a single intraperitoneal Injection of Aroclor 1254 (500 mg/kg body weight) diluted in Miglyol 812 oil (MERCK, Article-No. 6175). The animals received drinking water and Altromin standard diet ad libitum. About 16 hours before sacrifice, the feed was removed. Five to seven days after application of Aroclor, the rats were sacrificed and the livers collected in ice-cooled sterilized beakers containing 0.15 M KCI. The livers were homogenized in a sterile glass potter homogenizer containing 3 ml of 0.15 M KCI per gram of liver wet-weight. The homogenate was spun at 9000 x g for 10 minutes at about +4°C and the supernatant fluid was decanted and transferred into sterilized and precooled plastic tubes. The S9 was then frozen in liquid nitrogen and stored at -196°C.
- Composition and characterization of S9-Mix (quantity per mL S9-Mix):
1st series 2nd series
Liver homogenate (S9) 0.1 ml 0.2 mL
MgCI2/KCI aqueous solution (0.4 M/1.64 M) 0.02 mL 0.02 mL
Glucose-6-phosphate 1.5 mg 1.5 mg
NADP 3.15 mg 3.15 mg
Sodium phosphate buffer (0.2 M, pH 7.4) 0.5 mL 0.5 mL
Distilled water 0.38 mL 0.18 mL
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability). Every S9-batch is tested for its metabolic activity by the use of specific substrates, requiring different enzymes of the P450-isoenzyme family.

Components added to each plate:
Bacteria suspension 0.1ml
Test material (or solvent) 0.01-0.03 ml
Sodium phosphate buffer (in studies without metabolic activation) 0.5 ml
S9-Mix (in studies with metabolic activation) 0.5 ml
Top agar solution 2.0 ml
Test concentrations with justification for top dose:
The test material concentrations were used selected according to the EC and OECD guidelines for this test system and the requirements of the Labor Ministry of Japan:
1. Series: 5, 15.8, 50, 158, 500, 1580, and 5000 µg/plate
2. Series: 5, 15.8, 50, 158, and 500 µg/plate
Vehicle / solvent:
Acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
cumene hydroperoxide
other: Daunomycin, 2-Aminoanthracene
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) . triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation) (experiment 1); preincubation (experiment 2)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
none (experiment 1), 20 min (experiment 2)
- Exposure duration/duration of treatment:
2 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Revertant colonies were either scored using an Artek M 880 colony counter or manually. The presence of a background lawn of non-revertant cells was checked for each plate. Tables of individual and mean values are generated by use of a validated, automated data processing program
Evaluation criteria:
A test material is defined as non-mutagenic in this assay if "no" or "weak increases" occur in the first and second series of the main experiment. ("Weak increases" randomly occur due to experimental variation.) A test material is defined as mutagenic in this assay if: - a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;- "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.In all further cases, a third test series with the bacterial strain in question should be performed. If the criteria for a positive test result are not fulfilled in at least two out of the three series, the test material is defined as being non-mutagenic in this test system.
Statistics:
n.a.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: A weak precipitation of the test material on the agar plates was observed at concentrations > 50 µg/plate, a strong precipitation at concentrations > 500 µg/plate. Toxicity to the bacteria was not observed.


STUDY RESULTS
- Concurrent vehicle negative and positive control data
The negative control mutant frequencies were all in the regular range. Slight deviations from those values, if they occur, are accepted if they appear in only one test series and, furthermore, all other parameters of that series are in the regular range. The strain specific positive control compounds, namely daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine, and cumene hydroperoxide in the absence of S9-Mix, yielded the expected mutant frequencies that were greatly in excess of the negative controls. The genotype of the tester strains used was thus confirmed. 2-Aminoanthracene and benzo[a]pyrene, which require metabolic activation, were strongly mutagenic. This indicates that the exogenous metabolizing system used in the present investigation (S9-Mix) was functioning.


Ames test:
- Signs of toxicity : No signs of toxicity were observed (no thinning of the background lawn)
- Individual plate counts : please refer to attached pdf
- Mean number of revertant colonies per plate and standard deviation : please refer to attached pdf
Conclusions:
According to the criteria for negative and positive results predetermined in the Study Protocol the test item was not mutagenic with and without metabolic activation (S9 mix) under the described experimental conditions.
Executive summary:

The registered substance was tested for gene mutation according to OECD Guideline 471 following GLP.

Purpose

The purpose of this assay was to provide information on possible health hazards for the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in man.

Study Design

The investigations for the mutagenic potential of the test material were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed.

Results

The test material was dissolved in acetone and tested at concentrations ranging from 5 to 5000 µg/plate. Precipitation of the test material on the agar plates occurred at concentrations 500 µg/plate. Toxicity to the bacteria was not observed.

Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine and cumene hydroperoxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test material showed no increase in the number of revertants of any bacterial strain. According to the criteria for negative and positive results predetermined in the Study Protocol, the test material was not mutagenic under the described experimental conditions.

Conclusion

With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 DEC 2000 - 09 MAR 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was conducted according to Good Laboratory Practice (GLP) and followed the OECD Guideline for the testing of Chemicals: Genetic Toxicology: 473 In vitro mammalian chromosome aberration test.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9 after induction using Aroclor 1254
Test concentrations with justification for top dose:
0, 10.2, 20.5, 40.9, 81.9, 164, 328, 655, 1310, and 2620 µg/mL medium
Vehicle / solvent:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
+S9
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
-S9
Details on test system and experimental conditions:
According to guideline
Evaluation criteria:
According to guideline
Statistics:
Descriptive statistics
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Treatment of V79 cell cultures with the test material, in the absence and presence of S9 mix, did not increase the proportion of cells with aberrant chromosomes. Thus, the test material was not clastogenic in this in vitro test system.
Executive summary:

Purpose

The purpose of the in vitro chromosome aberration test is to identify agents that cause structural chromosome aberrations in cultured mammalian cells thus providing information on possible health hazards for the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in man.

Study Design

In this study the clastogenic potential of the test material was evaluated by examining its effects on the chromosomes of CHO cells, cultured in vitro and treated in the absence and presence of a rat liver metabolising system (S-9). The test methodology in this study is in accordance with current literature and complies with the OECD Guideline 473 (1997).

Results

The test material did not induce structural chromosome aberrations when tested at, or very close to, its limit of cytotoxicity following 24 hour treatment in the absence and presence of S9. No treatment-related increase of endoreduplications or polyploid cells was observed. Neither structural nor numerical aberrations were detected.

Conclusion

In conclusion, treatment of V79 cell cultures with the test material, did not increase the proportion of cells with aberrant chromosomes, thus the test material was not clastogenic in this in vitro test system.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 JUN 1998 - 11 DEC1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1997
Deviations:
no
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells:
L5178Y TK(+/-) mouse lymphoma cells. The original cultures were obtained from Dr. W. Muster, Hoffmann-La Roche, Basel, Switzerland on March 24, 1995.

For cell lines:
- Absence of Mycoplasma contamination:
yes, regularly checked
- Methods for maintenance in cell culture:
stored as frozen stocks in liquid nitrogen

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Growth media: Three media, supplementing RPMI 1640-medium with Glutamax 1 with different serum concentrations were used
Exposure medium: RPMI- 5 (RPM 1640 with 5% heat inactivated horse serum) 470 mL RPMI 164025 mL horse serum (heat-inactivated horse serum) 5 mL penicillin/streptomycin
Culture medium: RPMI-10 (RPMI 1640 with 10% heat-inactivated horse serum) 445 mL RPMI 164050 mL horse serum (heat-inactivated horse serum) 5 mL penicillin/streptomycin
Survivor- and selection medium: RPMI-20 (RPMI 1640 with 20% heat-inactivated horse serum) 395 mL RPMI 1640100 mL horse serum (heat-inactivated horse serum) 5 mL penicillin/streptomycin
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: 2% rat liver homogenate (S9 mix) with standard co-factors
- source of S9 : in-house, Institute of Toxicology of MERCK KGaA
- method of preparation of S9 mix : S9 is routinely prepared following the proposals of Ames et al. (1975). Instead of potassium chloride solution, as recommended by Ames, Dulbecco's phosphate buffered saline (PBS) which additionally contained 20 mM HEPES pH 7.4 (PBS-HEPES) is used.
- concentration or volume of S9 mix and S9 in the final culture medium: On the day of the experiment, gIucose-6-phosphate (590 mM), NADP (30 mM), KCI (150 mM) and rat liver S9 were mixed at the ratio of 1:1:1:2. For the treatment of the cells with test material, the final concentration of S9 in the cell culture medium was 2 %. For all cultures treated in the presence of S9 mix, a 1 mL aliquot of the mix was added to each cell culture (19 mL) to give a total of 20 mL. Cultures treated in the absence of S9 mix received 1 ml 150 mM KCI.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Every S9 batch is tested for its metabolic activity by the use of specific substrates, requiring different enzymes of the P450-isoenzyme family. The acceptance criterion was perfectly met.
Test concentrations with justification for top dose:
8.89, 28.1 and 88.9 µg/mL due to range-finding test, selected on the basis of the solubility and cytotoxicity characteristics
Vehicle / solvent:
Acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : single (duplicate for solvent control), screening version
- Number of independent experiments: 2 series

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 10E7
- Test substance added in medium: On day 1 of the experiment, at least 10E7 cells in a volume of 19 mL of RPMI 5 (to give a final concentration of 5 % serum) were placed in each of a series of sterile disposable 50 mL centrifuge tubes. Solvent, test article or positive control solution (0.2 mL if H2O is the solvent, 0.02 mL of organic solvents) and 1.0 mL of KCl (150 mM) or S9 mix were added.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 24 hours in the absence and 3 hours in the presence of S9 mix
After having been shaken for 3 hours in the presence and 24 hours in the absence of S9 mix at 37°C, the tubes were centrifuged at 188 x 'g' for 5 minutes, the cells washed with tissue culture medium and resuspended further in 10 ml RPMI 10 per tube. Cell densities were determined using a hemocytometer and the concentrations adjusted to 2 x 10E5/mL. Cells were transferred to flasks for growth through the expression period or were diluted to be plated for survival

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days (until day 3 of the experiment)
- Selection time (if incubation with a selective agent): From observations on recovery and growth of the cultures during the expression period, the cultures were selected to be plated for viability and TFT resistance (mutation assessment). Until scorable (day 7 to day 10).
- Method used: microwell plates for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.: 5-trifluorothymidine (TFT), final concentration 3 µg/mL
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 1 x 10E4/mL-> diluted to 8 cells/mL, staining with MTT, identified by eye and counted

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: relative total growth (RTG). In a preceding range finding test, cell growth was determined after exposure (3 hours in the presence and 24 hours in the absence of S9 mix) to various test material concentrations. The cell number was determined microscopically 24 hours after start of treatment and compared with cell number in the absence of the test material.
- Any supplementary information relevant to cytotoxicity: The test item was non-toxic in the range findeing test. Precipitation of the test item in the cell culture medium was macroscopically visible at the concentrations of 28.1 and 88.9 µg/mL.
Evaluation criteria:
Test materials are assessed as negative or non-mutagenic in this test system if
• the assay is considered valid and
• no relevant increase in the mutation frequency (at least a 2-fold) occurs.

Test materials are assessed as positive or mutagenic in this test system if
• the assay is considered valid and
• a clear increase in the mutation frequency (at least a 2-fold) occurs dose-dependently (over at least two test material concentrations) or reproducibly at identical concentrations in two independent experimental series performed.
In all other cases, further decisions for testing strategies should be made following the scientific evaluation of all existing data including those of non-toxicological investigations.
Statistics:
NA
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 88.9 µg/mL (without S9)
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: the test material precipitated in the cell culture medium at concentrations levels between 28.1 and 158 µg/ml.

RANGE-FINDING/SCREENING STUDIES (if applicable):
In a preceding range finding test, cell growth was determined after exposure (3 hours in the presence and 24 hours in the absence of S9 mix) to various test material concentrations. The cell number was determined microscopically 24 hours after start of treatment and compared with cell number in the absence of the test material. The substance was non-toxic in this range finding test. Precipitation in the cell culture medium was macroscopically visible at the concentrations of 28.1 and 88.9 µg/ml.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : In the absence and presence of S9 mix, the mutation frequency of the positive controls were clearly elevated.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: The test material was weakly toxic to the mouse lymphoma cells in the absence of S9 mix at the concentration of 88.9 µg/ml. No toxicity was seen in the presence of S9 mix.
Conclusions:
It is concluded that the test material is non-mutagenic in this screening test system under conditions where the positive controls exerted potent mutagenic effects.
Executive summary:

Purpose

The objective of this study was to evaluate the mutagenic activity of the test material by examining its ability to induce TK mutations in L5178Y cells in the absence and presence of a rat liver metabolizing system (S9 mix).

Study Design

The study was performed acording to OECD Guideline 473. The test material was screened for its ability to induce mutations at the TK locus (5-trifluorothymidine (TFT) resistance) in mouse lymphoma cells using a fluctuation protocol. The study was conducted in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pretreated with Aroclor 1254). The exposure time was 24 hours in the absence and 3 hours in the presence of S9 mix.

Results

The top doses to determine TFT resistance in this experiment were selected on the basis of the solubility and cytotoxicity characteristics of the test material. The test material was weakly toxic to the mouse lymphoma cells in the absence of S9 mix at the concentration of 88.9 µg/ml. Under the different experimental conditions of this study, the test material precipitated in the cell culture medium at concentration levels between 28.1 and 158 µg/ml. Concentrations ranging from 8.89 to 158 µg/ml were therefore tested.

Negative (solvent) and positive control treatments were included in each mutation experiment in the absence and presence of S9 mix. Mutant frequencies in negative control cultures fell within normal ranges, and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline N-oxide (without S9 mix) and benzo[a]pyrene (with S9 mix). Therefore the study was accepted as valid.

No relevant increases in mutant frequency were observed following treatment with the test material in neither the absence or presence of S9 mix.

Conclusion

It is therefore concluded that the test material is non-mutagenic in this screening test system under conditions where the positive controls exerted potent mutagenic effects.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

in vitro

Gene mutation in bacteria, OECD 471

The registered substance was tested for gene mutation according to OECD Guideline 471 following GLP.

Purpose

The purpose of this assay was to provide information on possible health hazards for the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in man.

Study Design

The investigations for the mutagenic potential of the test material were performed using Salmonella typhimuriumt ester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed.

Results

The test material was dissolved in acetone and tested at concentrations ranging from 5 to 5000 µg/plate. Precipitation of the test material on the agar plates occurred at concentrations 500 µg/plate. Toxicity to the bacteria was not observed.

Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine and cumene hydroperoxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test material showed no increase in the number of revertants of any bacterial strain. According to the criteria for negative and positive results predetermined in the Study Protocol, the test material was not mutagenic under the described experimental conditions.

Conclusion

With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

Chromosome aberration, OECD 473

Purpose

The purpose of the in vitro chromosome aberration test is to identify agents that cause structural chromosome aberrations in cultured mammalian cells thus providing information on possible health hazards for the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in man.

Study Design

In this study the clastogenic potential of the test material was evaluated by examining its effects on the chromosomes of CHO cells, cultured in vitro and treated in the absence and presence of a rat liver metabolising system (S-9). The test methodology in this study is in accordance with current literature and complies with the OECD Guideline 473 (1997).

Results

The test material did not induce structural chromosome aberrations when tested at, or very close to, its limit of cytotoxicity following 24 hour treatment in the absence and presence of S9. No treatment-related increase of endoreduplications or polyploid cells was observed. Neither structural nor numerical aberrations were detected.

Conclusion

In conclusion, treatment of V79 cell cultures with the test material, did not increase the proportion of cells with aberrant chromosomes, thus the test material was not clastogenic in this in vitro test system.

Mammalian gene mutation, OECD 476

The registered substance was tested for gene mutation according to OECD Guideline 476.

Purpose

The objective of this study was to evaluate the mutagenic activity of the test material by examining its ability to induce TK mutations in L5178Y cells in the absence and presence of a rat liver metabolizing system (S9 mix).

Study Design

The test material was screened for its ability to induce mutations at the TK locus (5-trifluorothymidine (TFT) resistance) in mouse lymphoma cells using a fluctuation protocol. The study was conducted in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pretreated with Aroclor 1254). The exposure time was 24 hours in the absence and 3 hours in the presence of S9 mix.

Results

The top doses to determine TFT resistance in this experiment were selected on the basis of the solubility and cytotoxicity characteristics of the test material. The test material was weakly toxic to the mouse lymphoma cells in the absence of S9 mix at the concentration of 88.9 µg/ml. Under the different experimental conditions of this study, the test material precipitated in the cell culture medium at concentration levels between 28.1 and 158 µg/ml. Concentrations ranging from 8.89 to 158 µg/ml were therefore tested.

Negative (solvent) and positive control treatments were included in each mutation experiment in the absence and presence of S9 mix. Mutant frequencies in negative control cultures fell within normal ranges, and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline N-oxide (without S9 mix) and benzo[a]pyrene (with S9 mix). Therefore the study was accepted as valid.

No relevant increases in mutant frequency were observed following treatment with the test material in neither the absence or presence of S9 mix.

Conclusion

It is therefore concluded that the test material is non-mutagenic in this screening test system under conditions where the positive controls exerted potent mutagenic effects.

Overall conclusion

The test material was not mutagenic in assays in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 and in E. coli wp2 in the presence or absence of exogenous metabolic activation system.

Treatment of V79 cell cultures with the test material, did not increase the proportion of cells with aberrant chromosomes, thus the test material was not clastogenic.

The test material was also non-mutagenic in mouse lymphoma cells with and without metabolic activation.

Justification for classification or non-classification

The key studies indicate no genotoxic potential present in vitro, thus no classification for mutagenicity is triggered in accordance with Regulation (EC) No 1272/2008.