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EC number: 285-089-6 | CAS number: 85029-63-6
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
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- Water solubility
- Solubility in organic solvents / fat solubility
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- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
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- Toxicological Summary
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Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
No mutagenicity of the test item was observed in a reliable in vitro gene mutation test in bacteria.
The test item did also not induce gene mutations in a reliable study using CHO cells in vitro with and without metabolic activation.
It was also negative in a reliable clastogenicity test in human lymphocytes in vitro with and without metabolic activation.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2004
- 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
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- The histidine dependent strains are derived from S. typhimurium strain LT2 throught a mutation in the histidine locus. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98 and TA 100 the R-factor plasmid pKM 101 carries the ampicillin resistance marker.
Strain WP2 and its derivatives all carry the same defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent (Trp+) mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagen which substitute one base for another. Additionally, the uvrA derivative is deficient in the DNA repair process (excision repair damage). Such a repair-deficient strain may be more readily mutated by agents.
When summarised the mutations on the TA strains and the E. coli strain, used in this study can be described as follows:
SALMONELLA TYPHIMURIUM:
-Strain TA 1537; Genotype: his C 3076; rfa-; uvrB-: Type of mutations indicated: frame shift mutations
-Strain TA 98; Genotype: his D 3052; rfa-; uvrB-; R-factor Type of mutations indicated: frame shift mutations
-Strain TA 1535; Genotype: his G 46; rfa-; uvrB-: Type of mutations indicated: base-pair substitutions
-Strain TA 100; Genotype: his G 46; rfa-; uvrB-; R-factor Type of mutations indicated: base-pair substitutions
ESCHERICHIA COLI
-Strain WP2 uvrA; Genotype: trp-; uvrA-: Escherichia coli: base-pair substitutions and others - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Test concentrations with justification for top dose:
- In the pre-experiment the concentration rnge of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I since no relevant toxic effects were observed and 5000 µg/plate were chosen as maximal concentration.
The concentration range included two logarithmic decades. The following concentrations were tested:
Pre-Experiment and Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate - Vehicle / solvent:
- On the day of the experiment, the test item was dissolved in DMSO. The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
The test item precipitated in the overlay agar in experiment I with and without metabolic activation at 5000, except in strain TA 1537 (without metabolic activation) and WP2 uvrA with metabolic activation precipitation was observed at 2500 - 5000 µg/plate.
The undissolved particles had no influence on the data recording. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 4-nitro-o-phenylene-diamine, 4-NOPD
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene, 2-AA
- Details on test system and experimental conditions:
- Negative Controls: Concurrent untreated and solvent controls were performed.
S9 (Preparation by RCC-CCR): Phenobarbital/beta-Naphthoflavone induced rat liver S9 is used as the metabolic activation system. The S9 is prepared from 8 - 12 weeks old male Wistar Hanlbm rats, weight approx. 220 - 320 g induced by applications of 80 mg/kg b.w. Phenobarbital i. p. (Desitin; D-22335 Hamburg) and beta-Naphthoflavone p.o. (Aldrich, D-89555 Steinheim) each on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCl solution followed by centrifugation at 9000 g. Aliquotes of the supernatant are frozen and stored in ampoules at -80 °C. Small numbers of the ampoules can be kept at -20 °C for up to one week.
The protein concentration in the S9 preparation was 34.5 mg/mL (lot no. R 300404).
S9 Mix:
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15 % v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al. - Rationale for test conditions:
- The experiments were performed to assess the potential of the test item to induce gene mutations by means of two independent Salmonella typhimurium and Escherichia coli reverse mutation assays. Experiment I was performed as a plate incorporation assay. Since a negative result was obtained in this experiment, experiment II was performed as a pre-incubation assay.
- Key result
- 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
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 2500 - 5000 µg/plate with and without S9 mix in Experiment I and at 33 - 1000 µg/plate without S9 mix and 2500 µg/plate with S 9 mix in Experiment II
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5000 µg/plate with S9 mix in Experiment I and II
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 1000 - 5000 µg/plate without S9 mix and at 2500 - 5000 µg/plate with S9 mix in Experiment I, at 333 - 5000 µg/plate without S9 mix and 2500 - 5000 µg/plate with S9 mix in Experiment II
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5000 µg/plate with and without S9 mix in Experiment I
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore the substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay. - Executive summary:
This study was performed to investigate the potential of the test substance to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.
The assay was performed in three independent experiments two with and without liver microsomal activation. A third experiment had to be performed, since in experiment I with metabolic activation in strain TA 1537 no bacteria were applicated onto the plates (reported as part of experiment I). Each concetration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:
Pre-Experiment and Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Reduced background growth was observed at the following concentrations (µg/plate):
Strain Experiment I Experiment II without S9 mix with S9 mix without S9 mix with S9 mix TA 1535 5000 5000 / / TA 1537 2500 - 5000 / 1000 2500 - 5000 TA 98 5000 2500 - 5000 5000 2500 - 5000 TA 100 333 - 5000 1000 - 5000 1000 - 5000 / WP2 uvrA 5000 1000 - 5000 / / / = no reduced background level
Toxic effects, evident as a reduction in the number of revertants, were observed at higher concentrations in strains TA 1537, TA 98, 100 and WP2 uvrA.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, meither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 13 May 2016 to 25 October 2016
- 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)
- Version / remarks:
- In accordance with the OECD guidelines for testing of chemicals, No. 473, “In Vitro Mammalian Chromosome Aberration Test” adopted on 26 September 2014.
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: In vitro Mammalian Chromosome Aberration Test
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- batch No.of test material: ESD0021912
- Expiration date of the batch: 17.03.2020
- Purity test date: 100% [w/w]
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29 °C) - Target gene:
- Not applicable
- Species / strain / cell type:
- lymphocytes:
- Details on mammalian cell type (if applicable):
- Human
- Additional strain / cell type characteristics:
- not applicable
- Cytokinesis block (if used):
- Colchicine
- Metabolic activation:
- with
- Metabolic activation system:
- Rat
- Test concentrations with justification for top dose:
- Based on the results of solubility, precipitation and pH tests, an initial cytotoxicity test was conducted for the selection of test concentrations for the chromosome aberration test. Top dose: 0.25 mg/mL
- Vehicle / solvent:
- Dimethylsulfoxide (DMSO)- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test item soluble in DMSO at 200 mg/mL - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- DURATION
- Preincubation period: 48 hours
- Exposure duration: 3-6 hours and 20-24 hours
NUMBER OF REPLICATIONS: Three concentratrion of test item, each concentrations in Duplicates
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Pellet was mixed with 5 mL of freshly prepared 0.56% warm Potassium chloride. Cell suspension was incubated for 10 minutes at room temperature and later centrifuged at 1800 to 2000 rpm for 10 minutes. Supernatant was discarded and cell pellet was mixed with 2 mL of freshly prepared cold acetic acid:methanol fixative (1:3). Cells were incubated for 10 minutes at room temperature and later suspension was centrifuged at 2200 rpm for 10 minutes. The procedure was repeated twice by adding 2 mL of cold acetic acid:methanol fixative (1:3). Clean slides were stored in a beaker with distilled water and kept in the refrigerator before use.
Slides were stained using 5% Giemsa stain for 20 minutes.
NUMBER OF CELLS EVALUATED: 150 metaphase plates for each replicate
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 metaphase
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication: - Rationale for test conditions:
- OECD guidelines for testing of chemicals, No. 473, “In Vitro Mammalian Chromosome Aberration Test”
- Evaluation criteria:
- • All slides including vehicle control, treatment and positive controls of chromosome aberration test were coded before evaluation.
• The cells were evaluated for structural aberrations in 150 metaphase plates for each replicate.
• Cytotoxicity was determined by calculating percentage reduction in mitotic index (%) for all treated and vehicle control cultures were determined.
• Gaps were recorded separately and reported but generally not included in the total aberration frequency.
• At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative/vehicle control. The increase is dose-related when evaluated with an appropriate trend test - Statistics:
- Data (Percentage of cells with aberrations) was analyzed using SPSS Software version 22 for differences among solvent/vehicle control, positive control and test item groups using ANOVA following Dunnett’s test at a 95% level of confidence (p < 0.05).
- Key result
- Species / strain:
- lymphocytes:
- 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:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Water solubility: No
- Precipitation: precipitation was observed at 0.5, 1.0 and 2.0 mg/mL - Remarks on result:
- other: non-clastogenic
- Conclusions:
- Based on the results obtained, the test item, Hostastat FE 20 LIQ is considered as non-clastogenic at and up to the dose of 0.25 mg/mL, both in the presence and absence of metabolic activation under the test conditions.
- Executive summary:
The test item,Hostastat FE 20 LIQ, wasevaluated for chromosomal aberration in human lymphocytes, as per the OECD guideline for the testing of chemicals,No. 473 “In vitroMammalian Chromosome AberrationTest” adopted on26thSeptember2014.
Test item was soluble in DMSO at 200 mg/mL. Slight precipitation was observed at 0.25 mg/mL and thereafter precipitation was observed at the higher doses up to 2 mg/mL. No change in pH was observed at any of the concentrations tested up to 2 mg/mL. As there was slight precipitation at 0.25 mg/mL, the same was selected as the highest concentration for testing in the initial cytotoxicity test.
In the initial cytotoxicity test, the test item resulted in not greater than 25.50% reduction of Mitotic Index (MI) at the concentrations of0.0625, 0.125 and 0.25mg/mL both at short term (in the presence and absence of metabolic activation) and long term treatments (in the absence of metabolic activation). Hence, the above indicated concentrations were selected for the chromosome aberration test.
In the chromosomal aberration test, the cells were treated withthe test itemat the doses of0.0625, 0.125 and 0.25mg/mL as low, mid and high doses respectivelyusing DMSO as the vehicle. The treatment was carried out in duplicates for short term period (3 to 6 hours) both in the presence and absence of metabolic activation and long term (20 to 24 hours) in the absence of metabolic activation.
Concentration of 10 µg/mL ofCyclophosphamide Monohydrate (+S9) and 0.05µg/mLof Mitomycin-C (-S9 both for short term and long term) were used as positive controls. Cells were arrested at metaphase using 0.3 µg/mL of colchicine. The cells were harvested between 2 to 3 hours of cell cycle arrest and slides were prepared and stained using 5% Giemsa stain.
The results indicatedno statistically significant increase in the number of aberrated cells when compared with vehicle control at any of the concentrations tested.The mean percentage reduction in mitotic index observed at 0.25 mg/mL, the highest concentration tested was 25.73 in the presence of metabolic activation and 24.39 and 26.82 in the absence of metabolic activation for short and long term treatments, respectively.
The respective positive controls tested induced 6.7% to 7.0% of mean aberrated cells which was statistically significant. The mean percentage reduction in mitotic index was in the range of 22.80 to 30.71 when compared with the respective vehicle controls.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 02 May 2016 to 01 July 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- OECD guidelines for testing of chemicals, No. 476, “In vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes” adopted on 28 July 2015.
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: Gene mutation test in mammalian cells in vitro
- Target gene:
- HPRT
- Species / strain / cell type:
- mammalian cell line, other:
- Details on mammalian cell type (if applicable):
- CHO AA8 cells
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9 homogenate
- Test concentrations with justification for top dose:
- 0.25 mg/mL, Based on the precipitation and pH test, 0.25 mg/mL was chosen as the highest dose for the initial cytotoxicity test and no cytotoxicity was observed in the initial cytotoxicity test up to 0.25 mg/mL tested, 0.25 mg/mL was chosen as the highest concentration for the gene mutation test.
- Vehicle / solvent:
- DMSO
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- benzo(a)pyrene
- Details on test system and experimental conditions:
- CHO AA8 cells, Batch No. 5000062 procured from American Type Culture Collection (ATCC)
and Alpha Minimal Essential Medium (MEM) without ribonucleosides containing 10% Fetal Bovine Serum (FBS) and antibiotics (1% Penicillin and streptomycin) - Evaluation criteria:
- There are several criteria for determining a positive result, such as a concentration-related increase or a reproducible increase in mutant frequency. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results.
- Statistics:
- Data of mutant frequencies was analyzed for differences among vehicle control, treatment and positive control groups using SPSS Software version 22 at a 95% level (p<0.05) of significance.
- Key result
- Species / strain:
- mammalian cell line, other: CHO AA8 cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- The test item, Hostastat FE 20 LIQ resulted in mutant frequencies of 15.35 to 17.39 mutant colonies/106 cells in the presence of metabolic activation and 16.39 mutant colonies/106 cells in the vehicle control. In the absence of metabolic activation, mutant frequencies of 15.12 to 16.91 mutant colonies/106 cells were observed with 15.89 mutant colonies/106 cells in the vehicle control. There was no concentrated related or statistically significant increase in the mutant frequencies when compared with vehicle control at any of the tested concentrations.
- Conclusions:
- Based on the results obtained, the test item is considered as non-mutagenic at and up to the concentration of 0.25 mg/mL, both in the presence and absence of metabolic activation under the laboratory conditions tested.
- Executive summary:
The test item was evaluated for gene mutation in CHO AA8 cells, as per the OECD guideline for the testing of chemicals, No. 476 “In vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes” adopted on 28 July 2015. Based on the results of solubility, pH and precipitation test, the test concentrations of 0.03125, 0.0625, 0.125 and 0.25 mg/mL were tested in the initial cytotoxicity test using DMSO as the vehicle both in the presence and absence of metabolic activation (3 to 6 hours). In the initial cytotoxicity test, the Relative Survival of the tested concentrations up to 2 mg/mL was more than 20%, when compared with the respective vehicle controls, both in the presence and absence of metabolic activation. Hence, concentrations of 0.03125, 0.0625, 0.125 and 0.25 mg/mL were selected for the gene mutation test. In the gene mutation test, the cells were treated with test item, at four concentrations using DMSO as a vehicle in duplicates in the presence and absence of metabolic activation (3 to 6 hours). Positive controls, 3 µg/mL of Benzo(a)pyrene (+S9) and 1 µg/mL of 4 Nitroquinoline 1- oxide (-S9) were used for the gene mutation test. Cytotoxicity of the test item and positive controls was assessed by the determining the Adjusted Cloning Efficiency (ACE) and Relative Survival (RS). The test item, Hostastat FE 20 LIQ resulted in mutant frequencies of 15.35 to 17.39 mutant colonies/106 cells in the presence of metabolic activation at the different test concentrations tested and 16.39 mutant colonies/106 cells in the vehicle control. In the absence of metabolic activation, test item resulted in mutant frequencies of 15.12 to 16.91 mutant colonies/106 cells and 15.89 mutant colonies/106 cells in the vehicle control. There was no concentration related or statistically significant increase in the mutant frequencies at any of the test item concentrations when compared with vehicle control. Positive controls resulted in mutant frequencies of 140.27 to 142.49 mutant colonies/106 cells in the presence and absence of metabolic activation, respectively and were statistically significant when compared with the respective vehicle controls.
Referenceopen allclose all
The test item was assessed for its poetntail to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.
The assay was performed in three independent experiments two with and without liver microsomal activation. A third experiment had to be performed, since in experiment I with metabolic activation in strain TA 1537 no bacteria were applicated onto the plates (reported as part of experiment I). Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:
Pre-Experiment and Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Reduced background growth was observed at the following concentrations (µg/plate):
Strain | Experiment I | Experiment II | ||
without S9 mix | with S9 mix | without S9 mix | with S9 mix | |
TA 1535 | 5000 | 5000 | / | / |
TA 1537 | 2500 - 5000 | / | 1000 | 2500 - 5000 |
TA 98 | 5000 | 2500 - 5000 | 5000 | 2500 - 5000 |
TA 100 | 333 - 5000 | 1000 - 5000 | 1000 - 5000 | / |
WP2 uvrA | 5000 | 1000 - 5000 | / | / |
/ = no reduced background growth
Toxic effects, evident as a reduction in the number of revertants, were observed at the following concentrations (µg/plate):
Strain | Experiment I | Experiment II | ||
without S9 mix | with S9 mix | without S9 mix | with S9 mix | |
TA 1535 | / | / | / | / |
TA 1537 | 2500 - 5000 | 2500 - 5000 | 33 - 1000 | 2500 |
TA 98 | / | 5000 | / | 5000 |
TA 100 | 1000 - 5000 | 2500 - 5000 | 333 - 5000 | 2500 - 5000 |
WP2 uvrA | 5000 | 5000 | / | / |
/ = no toxic effects evident as a reduction in the number of revertants
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.
In strain WP2 uvrA of the second experiment without metabolic activation the historical range of positive controls was just not reached. This minor effect was judged to represent fluctuations. The threshold of two times the corresponding solvent control was exceeded by far (factor of 6.5), so the test was considered valid.
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
TABLE 1. SUMMARY OF PERCENTAGE MITOTIC INDEX FOR INITIAL CYTOTOXICITY TEST
Set No. |
Treatment |
Dose (mg/mL) |
Mitotic Index |
Mean Mitotic Index |
Mean Percentage Mitotic Index |
Percentage Reduction in Mitotic Index |
|
Replicate 1 |
Replicate 2 |
||||||
Set 1 (+S9) (3-6 hours) |
Vehicle control |
- |
0.0533 |
0.0499 |
0.0516 |
5.16 |
- |
Test item [Hostastat FE 20 LIQ] |
0.0625 |
0.0455 |
0.0431 |
0.0443 |
4.43 |
14.03 |
|
0.125 |
0.0431 |
0.0406 |
0.0419 |
4.19 |
18.81 |
||
0.25 |
0.0394 |
0.0374 |
0.0384 |
3.84 |
25.50 |
||
|
|||||||
Set2 (-S9) (3-6 hours) |
Vehicle control |
- |
0.0544 |
0.0518 |
0.0531 |
5.31 |
- |
Test item [Hostastat FE 20 LIQ] |
0.0625 |
0.0438 |
0.0439 |
0.0439 |
4.39 |
17.36 |
|
0.125 |
0.0446 |
0.0418 |
0.0432 |
4.32 |
18.61 |
||
0.25 |
0.0400 |
0.0415 |
0.0408 |
4.08 |
23.23 |
||
|
|||||||
Set 3 (-S9) (20-24 hours) |
Vehicle control |
- |
0.0504 |
0.0524 |
0.0514 |
5.14 |
- |
Test item [Hostastat FE 20 LIQ] |
0.0625 |
0.0438 |
0.0456 |
0.0447 |
4.47 |
12.99 |
|
0.125 |
0.0411 |
0.0423 |
0.0417 |
4.17 |
18.88 |
||
0.25 |
0.0394 |
0.0375 |
0.0385 |
3.85 |
25.11 |
+S9: With metabolic activation; -S9: Without metabolic activation
TABLE 2. SUMMARY OFCHROMOSOMAL ABERRATIONSAND MITOTIC INDEX
Set No. |
Treatment |
Dose (mg/mL) |
Mean % MI |
Mean % Reduction in MI |
Mean of Total Aberrations with Gaps |
Mean of Total Aberrations without Gaps |
Mean of Total Aberrant cells without Gaps |
Mean of Percentage Aberrated Cells |
Set 1 (+S9) (3-6 hours) |
Vehicle control |
- |
3.89 |
- |
1.0 |
1.0 |
1.0 |
0.7 |
Positive Control (Cyclophosphamide) |
10 µg/mL |
3.17 |
22.80 |
13.0 |
11.0 |
10.5 |
7.0* |
|
Test item [Hostastat FE 20 LIQ] |
0.0625 |
3.44 |
14.39 |
2.5 |
1.5 |
1.5 |
1.0 |
|
0.125 |
3.28 |
19.29 |
1.5 |
1.0 |
1.0 |
0.7 |
||
0.25 |
3.08 |
25.73 |
3.5 |
2.5 |
2.5 |
1.7 |
MI: Mitotic Index; *: Statistically significant;+S9: With metabolic activation
TABLE 2 (Contd..,). SUMMARY OFCHROMOSOMAL ABERRATIONSAND MITOTIC INDEX
Set No. |
Treatment |
Dose (mg/mL) |
Mean % MI |
Mean % Reduction in MI |
Mean of Total Aberrations with Gaps |
Mean of Total Aberrations without Gaps |
Mean of Total Aberrant cells without Gaps |
Mean of Percentage Aberrated Cells |
Set 2 (-S9) (3-6 hours) |
Vehicle control |
- |
4.05 |
- |
1.0 |
1.0 |
1.0 |
0.7 |
Positive Control (Mitomycin-C) |
0.05 µg/mL |
3.26 |
24.42 |
13.0 |
10.5 |
10.5 |
7.0* |
|
Test item [Hostastat FE 20 LIQ] |
0.0625 |
3.48 |
17.66 |
2.5 |
1.5 |
1.5 |
1.0 |
|
0.125 |
3.42 |
19.44 |
2.5 |
1.5 |
1.5 |
1.0 |
||
0.25 |
3.26 |
24.39 |
2.5 |
2.0 |
2.0 |
1.3 |
MI: Mitotic Index; *: Statistically significant;-S9: Without metabolic activation
TABLE 2 (Contd..,). SUMMARY OFCHROMOSOMAL ABERRATIONSAND MITOTIC INDEX
Set No. |
Treatment |
Dose (mg/mL) |
Mean % MI |
Mean % Reduction in MI |
Mean of Total Aberrations with Gaps |
Mean of Total Aberrations without Gaps |
Mean of Total Aberrant cells without Gaps |
Mean of Percentage Aberrated Cells |
Set 3 (-S9) (20-24 hours) |
Vehicle control |
- |
4.26 |
- |
1.0 |
1.0 |
1.0 |
0.7 |
Positive Control (Mitomycin-C) |
0.05 µg/mL |
3.26 |
30.71 |
14.0 |
11.0 |
10.0 |
6.7* |
|
Test item [Hostastat FE 20 LIQ] |
0.0625 |
3.69 |
17.67 |
2.0 |
1.5 |
1.5 |
1.0 |
|
0.125 |
3.61 |
19.96 |
2.5 |
1.0 |
1.0 |
0.7 |
||
0.25 |
3.39 |
26.82 |
2.5 |
2.0 |
2.0 |
1.3 |
MI: Mitotic Index; *: Statistically significant;-S9: Without metabolic activation
TABLE 1. SUMMARY OF INITIAL CYTOTOXICITY TEST
Refer Appendix 1
Set No. |
Treatment |
Dose (mg/mL) |
Average colony count |
Adjusted Cloning Efficiency (ACE) |
Relative Survival (%) |
||||||
Set 1 +S9 |
Vehicle control |
- |
174.83 |
1.18 |
- |
||||||
Hostastat FE 20 LIQ |
0.03125 |
164.83 |
1.08 |
91.21 |
|||||||
0.0625 |
163.00 |
1.02 |
86.30 |
||||||||
0.125 |
164.50 |
1.01 |
85.97 |
||||||||
0.25 |
164.17 |
0.98 |
83.00 |
||||||||
|
|||||||||||
Set 2 -S9 |
Vehicle control |
-
|
171.83 |
1.13 |
- |
||||||
Hostastat FE 20 LIQ |
0.03125 |
171.00 |
1.11 |
97.99 |
|||||||
0.0625 |
167.67 |
1.07 |
95.18 |
||||||||
0.125 |
168.83 |
1.05 |
92.84 |
||||||||
0.25 |
166.00 |
1.02 |
90.69 |
+S9: with metabolic activation; -S9: without metabolic activation
TABLE 1. SUMMARY OF PARALLEL CYTOTOXICITY TEST- GENE MUTATION TEST
Refer Appendix2
Set No. |
Treatment |
Dose (mg/mL) |
Average colony count |
Adjusted Cloning Efficiency (ACE) |
Relative Survival (%) |
Set 1 +S9 |
Vehicle control |
-
|
179.67 |
1.14 |
- |
Hostastat FE 20 LIQ |
0.03125
|
174.50 |
1.10 |
96.79 |
|
0.0625
|
174.33 |
1.08 |
94.72 |
||
0.125
|
168.00 |
1.02 |
89.70 |
||
0.25
|
165.50 |
0.99 |
87.43 |
||
Benzo(a)pyrene |
0.003
|
148.17 |
0.79 |
69.35 |
|
|
|||||
Set 2 -S9 |
Vehicle control |
- |
176.83 |
1.12 |
- |
Hostastat FE 20 LIQ |
0.03125
|
175.67 |
1.10 |
98.34 |
|
0.0625
|
175.67 |
1.10 |
99.00 |
||
0.125
|
166.50 |
1.00 |
89.67 |
||
0.25
|
165.00 |
0.98 |
88.23 |
||
4 Nitroquinoline 1-oxide |
0.001 |
150.50 |
0.81 |
72.67 |
+S9: with metabolic activation; -S9: without metabolic activation.
TABLE 1. SUMMARY OF GENE MUTATION TEST
Refer Appendix 3
Set No. |
Treatment |
Dose (mg/mL) |
Average colony count |
Cloning Efficiency |
Average Mutant Colonies/ 106cells |
Mutant Frequency/ 106cells |
Set 1 +S9 |
Vehicle control |
- |
183.00 |
0.92 |
15.0 |
16.39 |
Hostastat FE 20 LIQ |
0.03125
|
172.50 |
0.86 |
14.0 |
16.22 |
|
0.0625
|
172.50 |
0.86 |
15.0 |
17.39 |
||
0.125
|
169.17 |
0.85 |
13.0 |
15.35 |
||
0.25
|
168.17 |
0.84 |
13.0 |
15.45 |
||
Benzo(a)pyrene |
0.003 |
165.33 |
0.83 |
115.5 |
140.27* |
|
|
||||||
Set 2 -S9 |
Vehicle control |
- |
188.67 |
0.94 |
15.0 |
15.89 |
Hostastat FE 20 LIQ |
0.03125
|
182.83 |
0.91 |
14.0 |
15.30 |
|
0.0625
|
171.83 |
0.86 |
13.0 |
15.12 |
||
0.125
|
164.83 |
0.82 |
13.0 |
15.80 |
||
0.25
|
165.67 |
0.83 |
14.0 |
16.91 |
||
4 Nitroquinoline 1-oxide |
0.001 |
160.00 |
0.80 |
114.0 |
142.49* |
+S9: with metabolic activation; -S9: without metabolic activation; *: Statistically significant
APPENDIX 1.INDIVIDUAL DATA OF INITIAL CYTOTOXICITY TEST
Set No. |
Treatment |
Dose (mg/mL) |
Replicate |
No. of Colonies |
||
Dish 1 |
Dish 2 |
Dish 3 |
||||
Set 1 +S9 |
Vehicle control |
- |
1 |
177 |
169 |
180 |
2 |
169 |
175 |
179 |
|||
Hostastat FE 20 LIQ |
0.03125 |
1 |
168 |
159 |
165 |
|
2 |
168 |
163 |
166 |
|||
0.0625 |
1 |
163 |
168 |
161 |
||
2 |
162 |
165 |
159 |
|||
0.125 |
1 |
163 |
169 |
162 |
||
2 |
164 |
166 |
163 |
|||
0.25 |
1 |
168 |
163 |
159 |
||
2 |
166 |
167 |
162 |
|||
Set 2 -S9 |
Vehicle control |
- |
1 |
176 |
173 |
168 |
2 |
169 |
166 |
179 |
|||
Hostastat FE 20 LIQ |
0.03125 |
1 |
171 |
176 |
170 |
|
2 |
168 |
168 |
173 |
|||
0.0625 |
1 |
167 |
165 |
170 |
||
2 |
171 |
164 |
169 |
|||
0.125 |
1 |
170 |
165 |
172 |
||
2 |
166 |
171 |
169 |
|||
0.25 |
1 |
165 |
164 |
169 |
||
2 |
168 |
167 |
163 |
+S9: with metabolic activation; -S9: without metabolic activation.
(Contd..,). INDIVIDUAL DATA OF INITIAL CYTOTOXICITY TEST
Set No. |
Treatment |
Dose (mg/mL) |
Replicate |
Cell count/ flask |
Average Cell count |
||
Set 1 +S9 |
End of treatment |
Vehicle Control |
1 |
4175000 |
4200000 |
||
2 |
4225000 |
||||||
Hostastat FE 20 LIQ |
0.03125 |
1 |
3975000 |
4062500 |
|||
2 |
4150000 |
||||||
0.0625 |
1 |
3900000 |
3887500 |
||||
2 |
3875000 |
||||||
0.125 |
1 |
3825000 |
3837500 |
||||
2 |
3850000 |
||||||
0.25 |
1 |
3750000 |
3712500 |
||||
2 |
3675000 |
||||||
Set 1 -S9 |
End of treatment |
Vehicle Control |
1 |
4125000 |
4087500 |
||
2 |
4050000 |
||||||
Hostastat FE 20 LIQ |
0.03125 |
1 |
4025000 |
4025000 |
|||
2 |
4025000 |
||||||
0.0625 |
1 |
4025000 |
3987500 |
||||
2 |
3950000 |
||||||
0.125 |
1 |
3850000 |
3862500 |
||||
2 |
3875000 |
||||||
0.25 |
1 |
3800000 |
3837500 |
||||
2 |
3875000 |
Beginning of treatment |
Replicate |
Cell count/ flask |
Average Cell count |
1 |
3100000 |
3112500 |
|
2 |
3125000 |
APPENDIX 1. INDIVIDUAL DATA OF PARALLEL CYTOTOXICITY TEST
Set No. |
Treatment |
Dose (mg/mL) |
Replicate |
No. of Colonies |
||
Dish 1 |
Dish 2 |
Dish 3 |
||||
Set 1 +S9 |
Vehicle control |
- |
1 |
180 |
182 |
178 |
2 |
176 |
179 |
183 |
|||
Hostastat FE 20 LIQ |
0.03125 |
1 |
177 |
169 |
181 |
|
2 |
169 |
170 |
181 |
|||
0.0625 |
1 |
177 |
176 |
173 |
||
2 |
176 |
174 |
170 |
|||
0.125 |
1 |
169 |
175 |
164 |
||
2 |
165 |
169 |
166 |
|||
0.25 |
1 |
167 |
164 |
163 |
||
2 |
170 |
165 |
164 |
|||
Benzo(a)pyrene |
0.003 |
1 |
156 |
149 |
146 |
|
2 |
147 |
143 |
148 |
|||
|
||||||
Set 2 -S9 |
Vehicle control |
- |
1 |
177 |
174 |
180 |
2 |
182 |
169 |
179 |
|||
Hostastat FE 20 LIQ |
0.03125 |
1 |
175 |
178 |
172 |
|
2 |
176 |
179 |
174 |
|||
0.0625 |
1 |
172 |
176 |
178 |
||
2 |
176 |
173 |
179 |
|||
0.125 |
1 |
170 |
169 |
164 |
||
2 |
168 |
166 |
162 |
|||
0.25 |
1 |
166 |
167 |
163 |
||
2 |
168 |
164 |
162 |
|||
4 Nitroquinoline 1-oxide |
0.001 |
1 |
150 |
142 |
154 |
|
2 |
155 |
150 |
152 |
+S9: with metabolic activation; -S9: without metabolic activation.
(Contd..,). INDIVIDUAL DATA OF PARALLEL CYTOTOXICITY TEST
Set No. |
Treatment |
Dose (mg/mL) |
Replicate |
Cell count/ flask |
Average Cell count |
||
Set 1 +S9 |
End of treatment |
Vehicle Control |
1 |
3650000 |
3687500 |
||
2 |
3725000 |
||||||
Hostastat FE 20 LIQ |
0.03125 |
1 |
3625000 |
3675000 |
|||
2 |
3725000 |
||||||
0.0625 |
1 |
3550000 |
3600000 |
||||
2 |
3650000 |
||||||
0.125 |
1 |
3500000 |
3537500 |
||||
2 |
3575000 |
||||||
0.25 |
1 |
3550000 |
3500000 |
||||
2 |
3450000 |
||||||
Positive Control |
1 |
3175000 |
3100000 |
||||
2 |
3025000 |
||||||
Set 1 -S9 |
End of treatment |
Vehicle Control |
1 |
3675000 |
3675000 |
||
2 |
3675000 |
||||||
Hostastat FE 20 LIQ |
0.03125 |
1 |
3550000 |
3637500 |
|||
2 |
3725000 |
||||||
0.0625 |
1 |
3700000 |
3662500 |
||||
2 |
3625000 |
||||||
0.125 |
1 |
3475000 |
3500000 |
||||
2 |
3525000 |
||||||
0.25 |
1 |
3425000 |
3475000 |
||||
2 |
3525000 |
||||||
Positive Control |
1 |
3100000 |
3137500 |
||||
2 |
3175000 |
Beginning of the treatment |
Replicate |
Cell count/ flask |
Average Cell count |
1 |
2950000 |
2912500 |
|
2 |
2875000 |
APPENDIX 1.INDIVIDUAL DATA OF GENE MUTATION TEST
Set No. |
Treatment |
Dose (mg/mL) |
No. of Colonies |
No. of Mutant Colonies |
||||||
Replicate 1 |
Replicate 2 |
Replicate 3 |
Replicate 1 |
Replicate 2 |
Replicate 3 |
Replicate 4 |
Replicate 5 |
|||
Set 1 +S9 |
Vehicle control |
- |
183 |
192 |
177 |
2 |
5 |
1 |
4 |
3 |
169 |
185 |
192 |
3 |
0 |
5 |
5 |
2 |
|||
Hostastat FE 20 LIQ |
0.03125 |
178 |
175 |
172 |
2 |
0 |
5 |
4 |
4 |
|
169 |
169 |
172 |
1 |
2 |
2 |
3 |
5 |
|||
0.0625 |
175 |
175 |
168 |
3 |
2 |
2 |
5 |
3 |
||
169 |
166 |
182 |
2 |
0 |
5 |
5 |
3 |
|||
0.125 |
175 |
162 |
175 |
0 |
5 |
2 |
4 |
4 |
||
175 |
166 |
162 |
2 |
3 |
2 |
0 |
4 |
|||
0.25 |
159 |
175 |
166 |
3 |
0 |
3 |
2 |
4 |
||
166 |
178 |
165 |
3 |
4 |
4 |
0 |
3 |
|||
Benzo(a)pyrene |
0.003 |
149 |
158 |
166 |
24 |
28 |
25 |
21 |
22 |
|
178 |
169 |
172 |
23 |
21 |
19 |
26 |
22 |
|||
|
||||||||||
Set 2 -S9 |
Vehicle control |
- |
189 |
196 |
189 |
3 |
4 |
4 |
2 |
3 |
178 |
192 |
188 |
3 |
2 |
1 |
4 |
4 |
|||
Hostastat FE 20 LIQ |
0.03125 |
182 |
169 |
188 |
3 |
2 |
2 |
5 |
1 |
|
179 |
187 |
192 |
3 |
2 |
4 |
1 |
5 |
|||
0.0625 |
166 |
178 |
178 |
2 |
3 |
5 |
0 |
4 |
||
169 |
175 |
165 |
2 |
1 |
2 |
4 |
3 |
|||
0.125 |
162 |
159 |
168 |
2 |
2 |
2 |
5 |
4 |
||
166 |
168 |
166 |
0 |
2 |
2 |
3 |
4 |
|||
0.25 |
163 |
163 |
169 |
4 |
4 |
2 |
2 |
3 |
||
169 |
168 |
162 |
2 |
2 |
2 |
4 |
3 |
|||
4 Nitroquinoline 1-oxide |
0.001 |
159 |
158 |
166 |
26 |
21 |
22 |
22 |
25 |
|
166 |
158 |
153 |
23 |
21 |
18 |
22 |
28 |
+S9: with metabolic activation; -S9: without metabolic activation.
APPENDIX 2. TEST ITEM AND POSITIVE CONTROL DILUTION PREPARATION FORGENE MUTATION TEST
Test Item |
Quantity of Test Item |
Concentration of Test Item Stock (mg/mL) |
Volume of Stock (µL) |
Volume of DMSO (µL) |
Concentration of Test Item Dilution (mg/mL) |
Treatment dose level (mg/mL) |
Hostastat FE 20 LIQ |
25 mg |
- |
- |
1000 |
25 |
0.25 |
- |
25 |
500 |
500 |
12.5 |
0.125 |
|
12.5 |
500 |
500 |
6.25 |
0.0625 |
||
6.25 |
500 |
500 |
3.125 |
0.03125 |
||
Positive Control |
Quantity of Positive Control |
Concentration of Stock (mg/mL) |
Volume of Stock (µL) |
Volume of DMSO (µL) |
Concentration of positive control dilution (mg/mL) |
Treatment dose level (mg/mL) |
|
2 mg |
- |
- |
2000 |
1000 |
- |
Benzo(a) Pyrene |
- |
1 |
300 |
700 |
300 |
3 |
|
2 mg |
- |
- |
2000 |
1000 |
- |
4 Nitroquinoline 1-oxide |
- |
1 |
100 |
900 |
100 |
1 |
Note: 50 µL of each test item dilutions, positive and vehicle control were added to each flask to a final volume of 5 mL.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Mode of Action Analysis / Human Relevance Framework
In the absence of any evidence for species specific effects or modes of action the possibly observed effects are regarded as relevant for humans.
Additional information
In the bacterial test, a plate incorporation test (experiment I) and a pre-incubation test (experiment II) were performed using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA in concentrations up to 5000 µg/plate with and without metabolic activation. No increase in revertants was observed. The study is GLP-compliant and was performed according to OECD guideline 471.
The test item was evaluated for gene mutation in CHO AA8 cells. Based on initial cytotoxicity testing, concentrations of 0.03125, 0.0625, 0.125 and 0.25 mg/mL were selected for the main test.
There was no concentration related or statistically significant increase in the mutant frequencies at any of the test item concentrations with and without metabolic activation. The study is GLP-compliant and was performed according to OECD guideline 476.
Clastogenicity in vitro was examined in a chromosomal aberration test with human lymphocytes. Based on a initial cyctotoxicity test, concentrations of 0.0625, 0.125 and 0.25mg/mL were tested for a short term period (3 to 6 hours) both in the presence and absence of metabolic activation and a long term period (20 to 24 hours) in the absence of metabolic activation. The results indicated no statistically significant increase in the number of aberrated cells at any of the concentrations tested. The study is GLP-compliant and was performed according to OECD guideline 473.
Justification for classification or non-classification
As no effects were observed in three reliable in vitro genotoxicity tests, no classification is required according to Regulation (EC) No. 1272/2008 (CLP).
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.