Registration Dossier
Registration Dossier
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 918-105-3 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
- Ames test: negative (+/- S9)
Read-across substance:
- Ames test: negative (+/- S9)
- in-vitro CA: positive (+/- S9)
- HPRT: negative (+/- S9)
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 17/0241-1, 294-296
- Expiration date of the lot/batch: 2020-05-09
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stable
- Stability under test conditions: yes - Target gene:
- histidine and tryptophan
- 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:
- S9 mix
- Test concentrations with justification for top dose:
- 0, 33, 100, 333, 1000, 2500, 5000µg/plate (SPT)
0, 33, 100, 333, 1000, 2500, 5000µg/plate (PIT, TA1535, TA100, TA98, E.coli WP2 uvrA, TA 1537 with S9)
0, 10, 33, 100, 333, 1000, 2500 µg/plate (PIT, TA1537 without metabolic activation)
In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- other: 2-aminoanthracene, N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylenediamine
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48-72h at 37°C in the dark
NUMBER OF REPLICATIONS: 3 - Evaluation criteria:
- Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used.
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other. - Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- In the SPT with metabolic activation cytotoxicity was observed at 5000µg/plate and in the PIT without metabolic activation from 2500 and with metabolic activation from 5000 µg/plate
- Vehicle 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:
- cytotoxicity
- Remarks:
- In the SPT cytotoxicity was observed from 5000µg/plate without metabolic activation and in the PIT from 2500 without and from 5000 µg/plate with metabloic activation.
- Vehicle 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:
- cytotoxicity
- Remarks:
- In the SPT cytotoxicity was observed from 1000µg/plate onwards and in the PIT from 33 µg/plate without and from 2500µg/plate onwards with metabolic activation.
- Vehicle 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:
- cytotoxicity
- Remarks:
- Cytotoxicty was observed in the SPT at 5000µg/plate and in the PIT without metabolic activation from 333µg/plate onwards and with metabolic activation at 5000µg/plate.
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Test substance precipitation of the test substance was found from about 1000 μg/plate onward with and without S9 mix in the standard plate test and without S9 mix in the preincubation test and from 2500 μg/plate with S9 mix in the preincubation test.
- Conclusions:
- Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.
- Executive summary:
The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay (TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA). The test concentrations were 33 μg - 5000 μg/plate (SPT) and 10 μg - 5000 μg/plate (PIT). Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was found depending on the strain and test conditions from about 1000 μg/plate onward. A bacteriotoxic effect was observed depending on the strain and test conditions from about 33 μg/plate onward. A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.
Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.
- 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
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- GLP compliance:
- yes (incl. QA statement)
- 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:
- phenobarbital/ß-naphthoflavone induced rat liver S9 mix
- Test concentrations with justification for top dose:
- Without S9 mix
Experiment IA: 19.5, 39.1, 78.1, 156.3, 312.5, 625.0, 1250.0, 2500.0, 5000.0 µg/mL
Experiment IB: 0.8, 1.6, 3.1, 6.3, 12.5, 25.0, 50.0, 100.0, 200.0 µg/mL
Experiment IC: 5.0, 10.0, 20.0, 30.0, 40.0, 50.0, 60.0, 75.0, 100.0 µg/mL
With S9 mix
Experiment IA: 19.5, 39.1, 78.1, 156.3, 312.5, 625.0, 1250.0, 2500.0, 5000.0 µg/mL
Experiment IC: 10.0, 20.0, 40.0, 60.0, 70.0, 80.0, 90.0, 100.0, 200.0 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- 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:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- Without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- With metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 14 hours
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa
NUMBER OF CELLS EVALUATED: 100
DETERMINATION OF CYTOTOXICITY
- Method: cell numbers - Evaluation criteria:
- A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the laboratory historical control data and
- no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations is not in the range of the laboratory historical control data and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of the laboratory historical control data. - Statistics:
- Statistical significance was confirmed by means of the Fisher’s exact test (7) (p < 0.05). However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- In Experiment IA, visible precipitation of the test item in the culture medium was observed at 78.1 µg/mL and above in the presence of S9 mix. In Experiment IB precipitation was observed at 50.0 µg/mL and above in the absence of S9 mix. In addition, precipitation occurred in Experiment IC at 75.0 µg/mL and above in the absence of S9 mix and at 100.0 µg/mL and above in the presence of S9 mix. No relevant influence on osmolarity or pH value was observed.
In Experiment IA and IB in the absence or presence of S9 mix concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In Experiment IC clear cytotoxicity indicated by reduced mitotic indices or cell numbers was observed at the highest evaluated concentrations (49.3 % and 49.2 % of control, respectively).
In Experiment IB and IC in the absence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 3.0 % aberrant cells, excluding gaps) were close to the range of the solvent control values (1.5 - 2.0 % aberrant cells, excluding gaps) and within the range of the laboratory historical solvent control data. In Experiment IA in the presence of S9 mix one statistically significant increase in chromosomal aberrations (6.0 % aberrant cells, excluding gaps) clearly exceeding the historical solvent control data (0.0 - 4.0 % aberrant cells, excluding gaps) was observed after treatment with 78.1 µg/mL. In the confirmatory experiment IC one statistically significant increase in chromosomal aberrations (7.5 % aberrant cells, excluding gaps) clearly exceeding the historical solvent control data was observed after treatment with 80.0 µg/mL. Thus the positive finding could be confirmed.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro. Therefore, the test item is considered to be clastogenic in this chromosome aberration test in the presence of metabolic activation, when tested up to cytotoxic, precipitating or the highest evaluable concentration..
- Executive summary:
The test item, dissolved in acetone, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in three independent experiments.
In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were evaluated for structural chromosome aberrations. The highest applied concentration (5000 Og/mL) was chosen with respect to the current OECD Guideline 473. Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of test item precipitation.
In Experiment IA and IB in the absence or presence of S9 mix concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In Experiment IC clear cytotoxicity indicated by reduced cell numbers or mitotic indices was observed at the highest evaluated concentrations. In the absence of S9 mix no clastogenicity was observed at the concentrations evaluated. In Experiment IA in the presence of S9 mix one statistically significant increase in chromosomal aberrations (6.0 % aberrant cells, excluding gaps) clearly exceeding the historical solvent control data (0.0 – 4.0 % aberrant cells, excluding gaps) was observed after treatment with 78.1 Og/mL. In the confirmatory experiment IC one statistically significant increase in chromosomal aberrations (7.5 % aberrant cells, excluding gaps) clearly exceeding the
historical solvent control data (0.0 – 4.0 % aberrant cells, excluding gaps) was observed after treatment with 80.0 Og/mL.
No evidence of an increase in polyploid metaphases was found after treatment with the test item as compared to the frequencies of the control cultures. Appropriate mutagens (EMS and CPA) were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- July - August 2012
- 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)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- HPRT (hypoxanthine-guanine phosphoribosyl transferase)
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- Before freezing, the level of spontaneous mutants was depressed by treatment with HAT-medium. Each batch is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency.
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/β-naphthoflavone induced rat liver S9 mix
- Test concentrations with justification for top dose:
- See any other information on materials and methods incl. tables.
- Vehicle / solvent:
- Solvent: acetone
The final concentration of acetone in the culture medium was 0.5 % (v/v). The solvent was chosen 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:
- ethylmethanesulphonate
- Remarks:
- Without metabolic activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 7,12-dimethylbenz(a)anthracene
- Remarks:
- With metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 24 hours
- Exposure duration: In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
- Expression/fixation time: Three or four days after treatment 1.5x10^6 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 - 5x10^5 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.
NUMBER OF REPLICATIONS: The study was performed in two independent experiments, using identical experimental procedures.
NUMBER OF CELLS EVALUATED: The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.
DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE). - Evaluation criteria:
- Acceptability of the Assay
The gene mutation assay is considered acceptable if it meets the following criteria:
The numbers of mutant colonies per 10^6 cells found in the solvent controls falls within the laboratory historical control data.
The positive control substances should produce a significant increase in mutant colony frequencies.
The cloning efficiency II (absolute value) of the solvent controls should exceed 50 %.
The data of this study comply with the above mentioned criteria.
Evaluation of Results
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 relevance and statistical significance was considered together.
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed in the first experiment at 30.0 µg/mL without metabolic activation and in the second experiment without metabolic activation precipitation occurred at 80.0 µg/mL. In both experiments in the presence of metabolic activation precipitation was observed at 40.0 µg/mL and above.
RESULTS GENOTOXICITY:
No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration. An increase of the induction factor exceeding the threshold of three times the mutation frequency of the corresponding solvent control was observed at almost all concentrations of the first culture of the first experiment without metabolic activation. This effect however was biologically irrelevant as it was based on the low solvent control of just 4.7 colonies per 10^6 cells and was neither dose dependent nor reproduced in the parallel culture under identical experimental conditions.
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 solely detected in the second culture of the second experiment with metabolic activation. This trend however, was judged as irrelevant since the absolute values of the mutation frequency remained within the historical range of solvent controls and the induction factor of 3.0 was not exceeded.
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 4.7 up to 29.8 mutants per 10^6 cells; the range of the groups treated with the test item was from 1.6 up to 48.3 mutants per 10^6 cells.
In the second culture of second experiment the cloning efficiency II of the solvent control without metabolic activation fell just short of the >50% limit of the acceptance criteria. The mean value of both parallel cultures however (0.6 and 0.5, equal to a mean of 0.55 or 55%) was acceptable.
EMS (150 μg/mL) and DMBA (1.1 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.
RANGE-FINDING/SCREENING STUDIES:
Based on the solubility properties of the test item the range finding pre-experiment test was performed using a concentration range of 19.6 to 2500 μg/mL to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation.
Relevant toxic effects were observed already at the lowest concentration of 19.6 μg/mL in the absence of metabolic activation after 4 hours treatment. At all higher concentrations the cell growth was completely inhibited. In the presence of metabolic activation (4 hours treatment) cytotoxic effects occurred at 156.3 μg/mL and above. Following continuous treatment (24 hours) toxic effects occurred at 78.1 μg/mL and above.
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 39.1 μg/mL and above in the absence of metabolic activation and at 78.1 μg/mL and above in the presence of metabolic activation. Following 24 hours treatment precipitation was determined at 78.1 μg/mL and above.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative cloning efficiency I or a relative cell density below 50 % were observed in the first experiment at 10.0 μg/mL and above without metabolic activation. In the presence of metabolic activation no cytotoxic effects were noted up to the second highest concentration of 80.0 μg/mL. At the maximum concentration of 160.0 μg/mL exceedingly severe cytotoxicity precluded evaluation of any results. In the second experiment cytotoxic effects as described above were noted at 80.0 μg/mL with and without metabolic activation.
The recommended cytotoxic range of approximately 10-20 % relative cloning efficiency I or relative cell density was covered with and without metabolic activation. - Conclusions:
- In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.
- Executive summary:
The study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. 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. The highest concentration applied in the pre-experiment (2500 μg/mL) was limited by the solubility properties of the test item in acetone and aqueous medium. The concentration range of the main experiments was limited by the cytotoxicity and precipitation of the test item. No substantial and reproducible dose dependent increase of the mutation frequency was
observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (EMS and DMBA), 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 at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.
Referenceopen allclose all
Standard Plate Test
E.coli WP2
| Dose [µg/plate] | Without S9 [Revertants/plate] |
With S9 [Revertants/plate] | ||
| Mean | Standard Deviation | Mean | Standard Deviation | |
| DMSO | 24.0 | 1.7 | 22.0 | 3.6 |
33 |
23.7 |
4.5 |
27.7 |
4.7 |
100 |
26.3 |
2.5 |
22.7 |
8.0 |
333 |
20.0 |
1.0 |
26.3 |
8.5 |
1000 |
21.7 |
1.2 |
24.3 |
5.1 |
2500 |
25.3 |
3.5 |
20.0 |
5.2 |
5000 |
19.7 |
7.5 |
21.7 |
2.1 |
5.0 (4 -NQO) |
740.0 |
51.1 |
- |
- |
60 (2 -AA) |
- |
- |
86.7 |
11.6 |
TA 1535
Dose [µg/plate] |
Without S9 [Revertants/plate] |
With S9 [Revertants/plate] |
||
|
Mean |
Standard Deviation |
Mean |
Standard Deviation |
DMSO |
9.3 |
3.2 |
8.7 |
3.1 |
33 |
12.0 |
0.0 |
10.0 |
4.4 |
100 |
10.7 |
1.5 |
10.3 |
1.2 |
333 |
9.0 |
4.4 |
7.7 |
0.6 |
1000 |
9.3 |
0.6 |
10.0 |
2.6 |
2500 |
7.3 |
0.6 |
8.7 |
1.5 |
5000 |
4.0 |
1.7 |
7.7 |
3.1 |
5.0 (MNNG) |
4514.0 |
374.4 |
- |
- |
2.5 (2 -AA) |
- |
- |
209.3 |
18.9 |
TA 100
Dose [µg/plate] |
Without S9 [Revertants/plate] |
With S9 [Revertants/plate] |
||
|
Mean |
Standard Deviation |
Mean |
Standard Deviation |
DMSO |
106.7 |
14.4 |
120.3 |
21.5 |
33 |
111.7 |
16.9 |
122.0 |
19.1 |
100 |
89.7 |
5.1 |
117.0 |
11.5 |
333 |
87.7 |
6.5 |
116.7 |
4.2 |
1000 |
95.7 |
16.2 |
122.0 |
10.5 |
2500 |
91.0 |
10.4 |
122.0 |
15.1 |
5000 |
66.3 |
4.0 |
95.0 |
10.4 |
5.0 (MNNG) |
3176.7 |
420.9 |
- |
- |
2.5 (2 -AA) |
- |
- |
1659.7 |
266.2 |
TA 1537
Dose [µg/plate] |
Without S9 [Revertants/plate] |
With S9 [Revertants/plate] |
||
|
Mean |
Standard Deviation |
Mean |
Standard Deviation |
DMSO |
8.3 |
2.3 |
7.3 |
4.0 |
33 |
8.7 |
3.5 |
7.7 |
3.8 |
100 |
7.3 |
3.2 |
5.7 |
2.1 |
333 |
7.7 |
4.7 |
7.3 |
5.8 |
1000 |
4.7 |
3.1 |
4.3 |
1.5 |
2500 |
3.7 |
1.2 |
4.7 |
0.6 |
5000 |
0.7 |
0.6 |
5.3 |
0.6 |
| 100 (AAC) | 763.3 | 36.9 | - | - |
| 2.5 (2 -AA) | - | - | 85.3 | 9.0 |
TA 98
Dose [µg/plate] |
Without S9 [Revertants/plate] |
With S9 [Revertants/plate] |
||
|
Mean |
Standard Deviation |
Mean |
Standard Deviation |
DMSO |
19.7 |
4.6 |
23.0 |
1.0 |
33 |
20.7 |
6.7 |
23.3 |
6.0 |
100 |
20.0 |
3.0 |
22.3 |
3.5 |
333 |
17.0 |
7.0 |
17.7 |
4.9 |
1000 |
17.3 |
3.1 |
25.3 |
10.2 |
2500 |
19.3 |
4.0 |
24.0 |
3.6 |
5000 |
9.7 |
0.6 |
12.7 |
5.1 |
10 (NOPD) |
922.0 |
56.6 |
- |
- |
2.5 (2 -AA) |
- |
- |
854.3 |
170.8 |
Preincubation Test
TA 1535
Dose [µg/plate] |
Without S9 [Revertants/plate] |
With S9 [Revertants/plate] |
||
|
Mean |
Standard Deviation |
Mean |
Standard Deviation |
DMSO |
8.0 |
3.0 |
9.7 |
1.5 |
33 |
9.0 |
7.2 |
6.7 |
2.1 |
100 |
10.7 |
5.7 |
7.7 |
2.1 |
333 |
8.0 |
4.6 |
8.0 |
4.4 |
1000 |
10.3 |
2.5 |
7.7 |
2.9 |
2500 |
3.7 |
2.1 |
8.0 |
3.6 |
5000 |
4.3 |
2.5 |
3.7 |
2.1 |
5.0 (MNNG) |
3529.7 |
196.4 |
- |
- |
2.5 (2 -AA) |
- |
- |
193.0 |
10.5 |
TA 100
Dose [µg/plate] |
Without S9 [Revertants/plate] |
With S9 [Revertants/plate] |
||
|
Mean |
Standard Deviation |
Mean |
Standard Deviation |
DMSO |
92.7 |
4.2 |
95.0 |
19.7 |
33 |
84.3 |
9.9 |
93.0 |
3.5 |
100 |
93.7 |
7.6 |
90.0 |
3.0 |
333 |
74.3 |
8.0 |
81.7 |
5.9 |
1000 |
81.0 |
18.0 |
86.0 |
12.5 |
2500 |
57.0 |
5.6 |
89.3 |
5.9 |
5000 |
33.0 |
19.9 |
60.3 |
9.0 |
5.0 (MNNG) |
2659.0 |
259.1 |
- |
- |
2.5 (2 -AA) |
- |
- |
1317.7 |
40.5 |
TA 1537
Dose [µg/plate]* |
Without S9 [Revertants/plate] |
With S9 [Revertants/plate] |
||
|
Mean |
Standard Deviation |
Mean |
Standard Deviation |
DMSO |
9.7 |
3.2 |
7.3 |
2.9 |
10 / 33 |
11.3 |
1.5 |
6.3 |
2.1 |
33 / 100 |
5.7 |
1.2 |
9.3 |
1.2 |
100 / 333 |
5.7 |
3.5 |
6.0 |
2.6 |
333 / 1000 |
3.3 |
2.5 |
6.3 |
1.5 |
1000 / 2500 |
3.3 |
1.5 |
4.3 |
2.9 |
2500 / 5000 |
1.0 |
1.0 |
3.3 |
1.5 |
100 (AAC) |
702.7 |
75.8 |
- |
- |
2.5 (2 -AA) |
- |
- |
59.7 |
10.4 |
* Test concentration without S9 on the left and with S9 on the right (-S9 / + S9)
TA 98
Dose [µg/plate] |
Without S9 [Revertants/plate] |
With S9 [Revertants/plate] |
||
|
Mean |
Standard Deviation |
Mean |
Standard Deviation |
DMSO |
17.3 |
3.8 |
22.7 |
2.5 |
33 |
12.7 |
3.1 |
18.0 |
4.6 |
100 |
16.7 |
3.8 |
19.7 |
2.1 |
333 |
9.3 |
2.1 |
17.3 |
6.7 |
1000 |
11.3 |
3.2 |
18.7 |
4.9 |
2500 |
8.0 |
1.7 |
17.3 |
5.0 |
5000 |
5.7 |
2.5 |
7.3 |
1.2 |
10 (NOPD) |
912.3 |
38.0 |
- |
- |
2.5 (2 -AA) |
- |
- |
952.7 |
18.1 |
E.coli WP2 uvrA
Dose [µg/plate] |
Without S9 [Revertants/plate] |
With S9 [Revertants/plate] |
||
|
Mean |
Standard Deviation |
Mean |
Standard Deviation |
DMSO |
19.7 |
6.1 |
20.0 |
1.0 |
33 |
19.0 |
6.0 |
23.0 |
4.0 |
100 |
22.3 |
4.2 |
18.7 |
7.6 |
333 |
19.3 |
1.5 |
18.7 |
4.0 |
1000 |
18.3 |
2.5 |
18.0 |
6.6 |
2500 |
22.0 |
4.6 |
17.3 |
4.0 |
5000 |
13.7 |
2.3 |
18.7 |
5.0 |
5.0 (4 -NQO) |
369.0 |
32.7 |
- |
- |
60 (2 -AA) |
- |
- |
77.0 |
13.5 |
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Genetic toxicity in vivo
Description of key information
Read-across substance:
- in-vivo MN: negative
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- GLP compliance:
- yes
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- NMRI
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: mean value 35.8 g (SD +- 1.9 g)
- Assigned to test groups randomly: [yes]
- Housing: single
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: for a minimum of five days after their arrival
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 2 °C
- Humidity (%): 45 - 77 %
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: [30% DMSO and 70% PEG 400]
- Justification for choice of solvent/vehicle: The vehicle was chosen due to its relative non-toxicity for the animals. - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test item was dissolved in 30% DMSO and 70% PEG 400. The vehicle was chosen due to its relative non-toxicity for the animals. All animals received a single standard volume orally.
Warming to 37°C and sonicating for a minimum of 15 minutes was used to formulate the test item. - Duration of treatment / exposure:
- single oral application
- Frequency of treatment:
- once
- Post exposure period:
- 24 or 48 hours
- Remarks:
- Doses / Concentrations:
500, 1000, and 2000 mg/kg bw
Basis:
actual ingested - No. of animals per sex per dose:
- 7 males for the test substance, 5 males for controls
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- cyclophosphamide
- Justification for choice of positive control(s):
- Route of administration potent inducer of micronuclei
- Doses / concentrations: 40 mg/kg bw. - Tissues and cell types examined:
- Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To investigate a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes.
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
A preliminary study on acute toxicity was performed with two animals per sex under identical conditions as in the mutagenicity study concerning: animal strain, vehicle, route, frequency, and volume of administration.
The animals were treated orally with the test item and examined for acute toxic symptoms at intervals of approximately 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item.
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animal’s body weight. The animals received the test item, the vehicle, or the positive control substance once orally. Seven males were treated per dose group and sampling time. Five males each were treated for each vehicle and the positive control group. The animals of all dose groups, except the positive control were examined for clinical signs at intervals of around 1 h, 2 - 4 h, 6 h, 24 h, and/or 48 h after administration of the test item and vehicles.
Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.
The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (Merck, 64293 Darmstadt, Germany)/Giemsa (Merck, 64293 Darmstadt, Germany). Cover slips were mounted with EUKITT (Kindler, 79110 Freiburg, Germany). At least one slide was made from each bone marrow sample.
DETAILS OF SLIDE PREPARATION and METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To investigate a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.
All animals per test group were evaluated as described. - Evaluation criteria:
- The study was considered valid as the following criteria are met:
- at least 5 animals per test group can be evaluated.
- PCE to erythrocyte ratio should not be less than 20 % of the vehicle control.
- the positive control shows a statistically significant and biological relevant increase of micronucleated PCEs compared to the vehicle control.
A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods (nonparametric Mann-Whitney test (8)) are used as an aid in evaluating the results, if necessary. However, the primary point of consideration is the biological relevance of the results.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system. - Statistics:
- Statistical methods (nonparametric Mann-Whitney test are used as an aid in evaluating the results, if necessary.
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
Two animals of each sex treated in the pre-experiment received the test item iGloss Crosslinker (ZQ54-2211) dissolved in 30% DMSO and 70% PEG 400 once orally. The volume administered was 10 mL/kg b.w..:
The animals treated with 2000 mg/kg b.w. (limit dose according to the guidelines) iGloss Crosslinker (ZQ54-2211) showed no clinical signs.
On the basis of these data the dose level 2000 mg/kg b.w. was estimated to be suitable as highest dose.
No substantial sex specific differences were observed with regard to clinical signs. In accordance with the test guidelines the main study was performed using males only.
RESULTS OF DEFINITIVE STUDY
In the main experiment for each test item dose groups 7 males received once orally administrations of iGloss Crosslinker (ZQ54-2211) dissolved in 30% DMSO and 70% PEG 400. The volume administered was 10 mL/kg b.w.. No clinical signs of toxicity were observed for all treated animals with the test item (dose levels 500, 1000 and 2000 mg/kg b.w.). The animals treated with the negative control (30% DMSO and 70% PEG 400) did not express any toxic reaction. - Conclusions:
- In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test item is considered to be non-mutagenic in this micronucleus assay.
- Executive summary:
This study was performed to investigate the potential of the test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was dissolved in 30% DMSO and 70% PEG 400, which was also used as vehicle control. The volume administered orally was 10 mL/kg b.w.. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis. Seven males per test group (except the vehicle and positive control groups with 5 males only) were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei. To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.
The following dose levels of the test item were investigated, based on results of a pre-experiment:
24 h preparation interval: 500, 1000, and 2000 mg/kg b.w.
48 h preparation interval: 2000 mg/kg b.w.
After treatment with the test item the number of PCEs per 2000 erythrocytes was not substantially decreased as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control thus indicating that the test item did not exert a cytotoxic effect in the bone marrow. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency.
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test item is considered to be non-mutagenic in this micronucleus assay.
Reference
The test item was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.
The test item was dissolved in 30% DMSO and 70% PEG 400, which was also used as vehicle control. The volume administered orally was 10 mL/kg b.w.. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.
Seven males per test group (except the vehicle and positive control groups with 5 males only) were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei.
To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.
The following dose levels of the test item were investigated, based on results of a pre-experiment:
24 h preparation interval: 500, 1000, and 2000 mg/kg b.w.
48 h preparation interval: 2000 mg/kg b.w.
The mean number of polychromatic erythrocytes (PCEs) per 2000 erythrocytes was not substantially decreased after treatment with the test item as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control indicating that iGloss Crosslinker (ZQ54-2211) did not induce cytotoxic effects in the bone marrow.
In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with the test item were below or near to the value of the vehicle control group and all values in all dose groups were very well within the historical vehicle control data range.
40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
in vitro:
Ames-Test
The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay (TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA). The test concentrations were 33 μg - 5000 μg/plate (SPT) and 10 μg - 5000 μg/plate (PIT). Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was found depending on the strain and test conditions from about 1000 μg/plate onward. A bacteriotoxic effect was observed depending on the strain and test conditions from about 33 μg/plate onward. A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.
Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.
The read-across substance was tested in a GLP compliant Ames reverse mutation assay according to OECD guideline 471 using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100 and Escherichia coli WP2 uvr A at 33 to 5000 µg/plate (two independent experiments (standard plate incorporation test and pre-incubation test) and each concentration was tested in triplicate) with and without metabolic activation (BASF SE, 2012). Precipitation of the test substance was found at 5000 μg/plate with and without S9 mix. A bacteriotoxic effect was observed depending on the strain and test conditions from about 2500 μg/plate onward. A relevant increase in the number of his+or trp+revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions of this study, the read-across substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.
Chromosome Aberration Test:
The read-across substance was tested in a GLP compliant in vitro mammalian chromosome aberration test according to OECD guideline 473 in which Chinese hamster V79 cells were exposed to 19.5 to 5000 (IA), 0.8 to 200 (IB) or 5 to 100 (IC) µg/mL without metabolic activation and 19.5 to 5000 (IA) or 10 to 200 (IC) µg/mL with metabolic activation (Harlan CCR, 2012).
In Experiment IA and IB in the absence or presence of S9 mix concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In Experiment IC clear cytotoxicity indicated by reduced cell numbers or mitotic indices was observed at the highest evaluated concentrations. In the absence of S9 mix no clastogenicity was observed at the concentrations evaluated. In Experiment IA in the presence of S9 mix one statistically significant increase in chromosomal aberrations (6.0 % aberrant cells, excluding gaps) clearly exceeding the historical solvent control data (0.0 – 4.0 % aberrant cells, excluding gaps) was observed after treatment with 78.1 µg/mL. In the confirmatory experiment IC one statistically significant increase in chromosomal aberrations (7.5 % aberrant cells, excluding gaps) clearly exceeding the historical solvent control data (0.0 – 4.0 % aberrant cells, excluding gaps) was observed after treatment with 80.0 µg/mL. No evidence of an increase in polyploid metaphases was found after treatment with the test item as compared to the frequencies of the control cultures. Appropriate mutagens (EMS and CPA) were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations. In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro. Therefore, the read-across substance is considered to be clastogenic in this chromosome aberration test in the presence of metabolic activation, when tested up to cytotoxic, precipitating or the highest evaluable concentration.
HPRT-Test:
A GLP-compliant gene mutation assay, tested according to OECD guideline 476, was performed to investigate the potential of the read-across substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster (Harlan 2012). The assay was performed in two independent experiments, using two parallel cultures each. The first 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.The concentration range of the main experiments was limited by the cytotoxicity and precipitation of the test item. Relevant cytotoxic effects were observed in the first experiment at 10.0μg/mL and above without metabolic activation. In the presence of metabolic activation no cytotoxic effects were noted up to the second highest concentration of 80.0μg/mL. At the maximum concentration of 160.0μg/mL exceedingly severe cytotoxicity precluded evaluation of any results. In the second experiment cytotoxic effects as described above were noted at 80.0μg/mL with and without metabolic activation. The recommended cytotoxic range of approximately 10-20 % relative cloning efficiency I or relative cell density was covered with and without metabolic activation. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Therefore, the read-across susbtance is considered to be non-mutagenic in this HPRT assay.
in vivo:
Micronucleus Assay
This study was performed to investigate the potential of the read-across substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse according to OECD 474 guideline and GLP (Harlan, 2013).
The test item was dissolved in 30% DMSO and 70% PEG 400, which was also used as vehicle control. The volume administered orally was 10 mL/kg b.w.. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.
Seven males per test group (except the vehicle and positive control groups with 5 males only) were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei.
To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.
The following dose levels of the test item were investigated, based on results of a pre-experiment:
24 h preparation interval: 500, 1000,
and 2000 mg/kg b.w.
48 h preparation interval: 2000 mg/kg b.w.
After treatment with the test item the number of polychromatic erythrocytes (PCEs) per 2000 erythrocytes was not substantially decreased as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control thus indicating that the read-across substance did not exert a cytotoxic effect in the bone marrow.
In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used.
40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency
In conclusion, it can be stated that under the experimental conditions reported, the test item the read-across substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.
Therefore, the read-across substance is considered to be non-mutagenic in this micronucleus assay.
Justification for classification or non-classification
Negative results were obtained with an Ames-Test of the registered substance and the read-across substance as well as with an HPRT-Test with the read-across substance. The positive in vitro results in a chromosome aberration test performed with the read-across substance could not be confirmed in an in vivo Micronucleus Assay. Therefore no classification for mutagenicity is required according to EU directive 67/548/EEC and EU classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
