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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.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: 233-149-7 | CAS number: 10045-86-0
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Bacterial mutagenicity: OECD 471, RL1: negative
Mammalian mutagenicity: OECD 476, RL1: negative
Cytogenicity: OECD 487, RL1: negative
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:
- November 10, 2015 until November 26, 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted July 21, 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- Regulation (EC) No. 440/2008, dated May 31, 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, Germany
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix (Aroclor 1254-induced)
- Test concentrations with justification for top dose:
- 31.6, 100, 316, 500, 2000 or 5000 μg
Two preliminary cytotoxicity tests (plate incorporation test, without and with metabolic activation) were carried out in test strain TA100.
5000 μg/plate was chosen as top concentration as no signs of cytotoxicity were noted. - Vehicle / solvent:
- - Vehicle used: 0.05 M HCl solution
- Justification for choice of vehicle: The test item was not soluble in any of the solvents recommended, dimethylsulfoxide (DMSO), ethanol, acetone or 0.05 M H2SO4 solution, therefore HCl was used. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- HCl solution
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- without metabolic activation: TA1535, TA100
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- HCl solution
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- Remarks:
- without metabolic activation: TA98
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- HCl solution
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- without metabolic activation: TA1537
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- HCl solution
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- without metabolic activation: TA102
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- HCl solution
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- with metabolic activation: TA98, TA102, TA1537
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-amino-anthracene
- Remarks:
- with metabolic activation: TA100, TA1535
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) and preincubation
DURATION
Preincubation Method:
- Preincubation period: 20 minutes at 37°C
In agar (plate incorporation)/ Preincubation method
- Exposure duration: 48 - 72 hours
NUMBER OF REPLICATIONS: 3 per concentration and experiment
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (colony counter) - Evaluation criteria:
- Mutagenicity
The test item is considered to show a positive response if:
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
- in addition, a significant (p ≤ 0.05) concentration (log value)-related effect (Spearman’s rank correlation coefficient) is observed;
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
Cytotoxicity
Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the vehicle control and/or a scarce background lawn. - Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5000 μg/plate without S9-mix
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- 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 5000 μg/plate with and without S9-mix
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- 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/ without S9-mix (only plate incorporation test)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- 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 5000 μg/plate in plate incorporation test without S9-mix
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5000 μg/plate in plate incorporation test with S9 mix
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Yes, test item precipitation was noted in both experiments, each carried out without and with metabolic activation, at concentrations of 316 μg/plate and higher in all test strains.
RANGE-FINDING/SCREENING STUDIES: Yes top concentration was determined to be 5000 μg/plate.
HISTORICAL CONTROL DATA (ranges, means and standard deviation and confidence interval (e.g. 95%) see table no.1)
- Positive historical control data: Valid, within the expected range
- Negative (vehicle) historical control data: Valid, within the expected range
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Colony counter
Cytotoxicity (reduction of the number of revertants by more than 50%) was noted in the following experiments at the top concentration of 5000 μg/plate:
Plate incorporation test:
without S9: in test strains TA98, TA100, TA1535 and TA1537;
with S9: in test strains TA98, TA102 and TA1537;
Preincubation test:
without S9 in test strains: TA1535 and TA1537
with S9 in test strains: TA98 and TA1537. - Conclusions:
- The test item was determined to be not mutagenic in the bacterial reverse mutation test with Salmonella typhimurium strains with and without metabolic activation.
- Executive summary:
In the GLP and OECD guideline 471 compliant bacterial reverse mutation study (Ames test) the test item was tested for mutagenicity in 5 Salmonella typhimurium strains.
Following strains were used TA98, TA100, TA102, TA1535 and TA1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test.
In the Preliminary test ranging from 0.316 to 5000 μg/plate the top concentration for the main study was determined to be 5000 μg/plate, where no signs of cytotoxicity were noted.
In the main study the test item was suspended in 0.05 M HCl solution for concentrations of 316, 500, 2000 or 5000 μg per plate. For concentrations lower than 100 μg/plate, the test item was completely dissolved. The vehicle 0.05 M HCl solution served as the negative control.
Test item precipitation was noted in both experiments, each carried out without and with metabolic activation, at concentrations of 316 μg/plate and higher in all test strains. In addition, cytotoxicity (reduction of the number of revertants by more than 50%) was noted in the following experiments at the top concentration of 5000 μg/plate:
Plate incorporation test:
without S9: in test strains TA98, TA100, TA1535 and TA1537;
with S9: in test strains TA98, TA102 and TA1537;
Preincubation:
without S9 in test strains: TA1535 and TA1537
with S9 in test strains: TA98 and TA1537
No increase in revertant colony numbers as compared with control counts was observed for the test item, tested up to concentration of 5000 μg/plate in any of the 5 test strains in two independent experiments without and with metabolic activation. The positive control items showed a significant increase in the number of colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- November 10, 2015 until December 15, 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- 2014
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, Germany
- Type of assay:
- in vitro mammalian cell micronucleus test
- Target gene:
- not applicable
- Species / strain / cell type:
- lymphocytes: human peripheral
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Human
- Sex, age and number of blood donors:male or female individuals, 18 – 35 years of age, healthy, non-smoking with no known recent exposures to genotoxic chemicals or radiation
- Whether whole blood or separated lymphocytes were used: Small innocula of whole blood (0.5 mL)
- Number of passages if applicable: Time equivalent to about 1.5 times the normal
MEDIA USED
- Type and identity of media: Fresh Ham’s F10 medium (1) with fetal calf serum (FCS) (2)
1) GIBCO Invitrogen GmbH, Technologiepark Karlsruhe, 76131 Karlsruhe, Germany
2) SIGMA-ALDRICH Chemie GmbH, 82024 Taufkirchen, Germany - Additional strain / cell type characteristics:
- not specified
- Cytokinesis block (if used):
- Yes
- Metabolic activation:
- with and without
- Metabolic activation system:
- Post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254
- Test concentrations with justification for top dose:
- Without and with metabolic activation (4-h exposure): 0.3162, 1.0, 3.162 or 10 μg/mL
Without metabolic activation (24-h exposure): 1.0, 3.162 or 10 μg/mL
Top dose was determined in preliminary cytotoxicity test without and with metabolic activation with concentrations of 0.01, 0.03162, 0.1, 0.3162, 1.0, 3.162 or 10.0 μg/mL. - Vehicle / solvent:
- - Vehicle used: 0.05 M HCl
- Justification for choice of vehicle: The test item was not soluble in any of the solvents recommended: aqueous media, dimethylsulfoxide (DMSO), ethanol, acetone or 0.05 M H2SO4 solution. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- 0.05 M HCl
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- other: Colchicine
- Remarks:
- without S9 mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- 0.05 M HCl
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- with S9 mix
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: In medium
DURATION
- Exposure duration: 4 hours and 24 hours (without S9-mix); 4-hours (with S9-mix)
- Selection time: 20 hours
- Fixation time: 24 hours (4-h exposure); 44 hours (24-h exposure)
SELECTION AGENT: 5 μg/mL Cytochalasin B (CytoB)
STAIN: 10% Giemsa
NUMBER OF REPLICATIONS: Two replicate cultures were used for each test item concentration and for the vehicle and positive control cultures.
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
After the incubation with the test item and with CytoB, the cells were fixed with freshly prepared fixative (3 parts methanol : 1 part glacial acetic acid v/v).
Two drops of this cell suspension were dropped onto a prewarmed, pre-cleaned microscope slide and left to air-dry at room temperature.
The slides were then stained using 10% Giemsa.
NUMBER OF CELLS EVALUATED: 500 cells
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: The micronucleus frequencies were analysed in at least 2000 binucleated cells per concentration (at least 1000 binucleated cells per culture; two cultures per concentration).
Only the frequencies of binucleate cells with micronuclei (independent of the number of micronuclei per cell) were used in the evaluation of micronucleus induction.
Care was taken not to score binucleate cells with irregular shapes or where the two nuclei differ greatly in size; neither would binucleate cells be confused with poorly spread multi-nucleate cells. Cells containing more than two main nuclei were not analysed for micronuclei, as the baseline micronucleus frequency might be higher in these cells.
DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis-Block Proliferation Index (CBPI) or the Replicative Index (RI) - Evaluation criteria:
- ACCEPTABILITY CRITERIA
Acceptance of a test is based on the following criteria:
- The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database (Poisson-based 95% control limits). Where concurrent negative control data fall outside the 95% control limits, they may be acceptable for inclusion in the historical control data as long these data are not extreme outliers.
- Concurrent positive controls induce responses that are compatible with those generated in the laboratory’s historical positive control data base and produce a statistically significant increase compared with the concurrent negative control.
- Adequate number of cells, cell proliferation criteria and concentrations are analysable and are consistent with those described.
Where cytotoxicity occurs, the test concentrations selected should cover a range from that producing 55 ± 5% cytotoxicity, to little or no cytotoxicity.
Vehicle control and untreated cultures give reproducibly low and consistent micronuclei frequencies. Data from vehicle and positive controls are used to establish historical control ranges. These values are used in deciding the adequacy of the concurrent vehicle controls or positive controls for an experiment.
INTERPRETATION OF RESULTS
A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
- the increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical negative control data (Poisson-based 95% control limits)
When all of these criteria are met, the test chemical is then considered able to induce chromosome breaks and/or gain or loss in this test system. - Statistics:
- Significance: CHI-SQUARE Test
- Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Yes, at 10.0 μg/mL
- Definition of acceptable cells for analysis: Binucleate cells
RANGE-FINDING/SCREENING STUDIES:
Test item precipitation was noted at a concentration of 10.0 μg/mL (24-hour or 4-hour exposure) in the experiment without and with metabolic activation. No signs of cytotoxicity were noted. Hence, 10.0 μg/mL were employed as the top concentration for the genotoxicity tests without and with metabolic activation.
CYTOKINESIS BLOCK
- Distribution of mono-, bi- and multi-nucleated cells: No data
NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: 5.5 to 7.0 micronucleated cells per 1000 binucleated cells (with S9-mix);
4.0 to 7.0 micronucleated cells per 1000 binucleated cells (without S9-mix)
HISTORICAL CONTROL DATA
- Positive historical control data: Valid (see table no.4)
- Negative (solvent) historical control data: Valid (see table no.4)
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Cytokinesis-Block Proliferation Index (CBPI) or the Replicative Index (RI)
- Other observations: frequency of binucleated cells - Conclusions:
- The test item revealed no indications of chromosomal damage in the in vitro micronucleus test in the absence and in the presence of metabolic activation, under the conducted test conditions.
- Executive summary:
In a GLP and guideline compliant study the test item was tested in an in vitro micronucleus test using human peripheral lymphocytes.
The test was carried out employing 2 exposure times without S9 mix: 4 and 24 hours, and 1 exposure time with S9 mix: 4 hours. The harvesting time was 20 hours after the end of exposure. The cytokinesis-block technique was applied. The test item was suspended in 0.05 M HCl solution for a concentration of 10 μg per mL medium. For concentrations lower than 10 μg/mL medium, the test item was completely dissolved. The vehicle 0.05 M HCl solution served as the negative control. In a preliminary experiment 10.0 μg/mL were employed as the top concentration for the genotoxicity tests without and with metabolic activation.
In the main study test item precipitation was noted in the experiments without and with metabolic activation at the top concentration of 10.0 μg test item/mL medium. No signs of cytotoxicity were noted. Mitomycin C (at 0.2 μg/mL) and Colchicine (at 0.02 μg/mL) were employed as positive controls in the absence and Cyclophosphamide (at 20 μg/mL) in the presence of metabolic activation.
The cells were treated with the test item concentrations of 0.3162, 1.0, 3.162 or 10.0 μg/mL. There was no dose-related increase in micronuclei up to the top concentration of 10.0 μg/mL neither in the tests without metabolic activation (4- and 24-hour exposure) nor in the test with metabolic activation (4-hour exposure). The frequency of micronucleated cells was within the historical control range of the untreated and vehicle controls.
The test item revealed no indications of chromosomal damage in the in vitro micronucleus test in the absence and in the presence of metabolic activation, under the conducted test conditions.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- The experimental phases of the study were performed between 26 May 2011 and 25 July 2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- July 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Version / remarks:
- Guideline 490 was not available when the study was performed.
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Target gene:
- Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media:
RPMI 1640
- Properly maintained:
yes
- Periodically checked for Mycoplasma contamination:
yes
- Periodically checked for karyotype stability:
no
- Periodically "cleansed" against high spontaneous background:
yes - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbital and beta-naphthoflavone induced rat liver S9 fraction
- Test concentrations with justification for top dose:
- the maximum dose level was (10mM) 1508 µg/ml
Vehicle and positive controls were used in parallel with the test item. Solvent (R0) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS), Sigma batch 0001423147 at 400 µg/ml and 150 µg/ml for Experiment 1 and Experiment 2 respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0164185 at 2 µg/ml was used as the positive control in the presence of metabolic activation - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used:
Solvent (R0) treatment groups were used as the vehicle controls.
- Justification for choice of solvent/vehicle:
Formed the best doseable suspension - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Solvent (R0 medium) treatment groups were used as the vehicle controls.
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- With metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Solvent (R0 medium) treatment groups were used as the vehicle controls.
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- Without metabolic activation.
- Details on test system and experimental conditions:
- The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese EPA/METI/MHLW guidelines for testing of new chemical substances.
Methods. Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-Hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at six dose levels using a 4 Hour exposure group in the presence of metabolic activation (1% S9) and a 24 Hour exposure group in the absence of metabolic activation.
The dose range of test item was selected following the results of a preliminary toxicity test and for the first experiment was 23.56 to 1508 µg/ml in the absence and presence of metabolic activation. For the second experiment the dose range was 94.25 to1508 µg/ml in the absenceand presence of metabolic activation.
The maximum dose level used in the mutagenicity test was the maximum recommended dose (10mM) of 1508 µg/ml. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. - Evaluation criteria:
Please see ''any other information on materials and methods incl. tables'' section.- Statistics:
- Please see ''any other information on materials and methods incl. tables'' section.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Remarks:
- Thymidine Kinase TK+/-
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- non-mutagenic
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- The dose range of the test item used in the Preliminary Toxicity Test was 5.89 to 1508 µg/ml
A precipitate was observed in all test item dose levels at the end of the exposure periods. Moderate reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item were observed in the 4-Hour exposure groups both in the absence and presence of metabolic activation when compared to the concurrent vehicle control groups. No marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item were observed in the 24-Hour exposure group when compared to the concurrent vehicle control group. Because of the reduction in %RSG observed in the 4-hour cultures with and without S9 the maximum dose level selected for the mutagenicity test was 1508 µg/ml, the 10 mM limit dose level
Mutagenicity Test
A summary of the results from the test is presented in attached in Table 1
Experiment 1
The results of the microtitre plate counts and their analysis are presented in Tables 2 to 7.
As was seen in the preliminary toxicity test a precipitate was observed in all test item dose levels and in both exposure groups at the end of the exposure period.
There was evidence of moderate test item-induced toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the %RSG values (Tables 3 and 6). There was no evidence of reductions in viability (%V) in both the absence and presence of metabolic activation, indicating that residual toxicity had not occurred (Tables 3 and 6). Acceptable levels of toxicity were seen with both positive control substances (Tables 3 and 6).
Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 3 and 6).
The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 3 and 6). A precipitate of test item was observed at and above 23.56 µg/ml.
The numbers of small and large colonies and their analysis are presented in Tables 4 and 7.
Experiment 2
The results of the microtitre plate counts and their analysis are presented in Tables 8 to 13.
As was seen in the preliminary toxicity test a precipitate was observed in all test item dose levels and in both exposure groups at the end of the exposure period.
As was seen previously, there was evidence of moderate toxicity in both the absence and presence of metabolic activation following exposure to the test item, as indicated by the %RSG values (Tables 9 and 12). There was no evidence of reductions in viability (%V) in both the absence and presence of metabolic activation, indicating that no residual toxicity had occurred (Tables 9 and 12). Acceptable levels of toxicity were seen with both positive control substances (Tables 9 and 12).
The 24-Hour exposure without metabolic activation demonstrated that the extended time point had no marked effect on the toxicity of the test item.
Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 9 and 12).
The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 9 and 12). All test item mutation values were within the acceptable range for vehicle control cultures. A precipitate of test item was observed at and above 94.25 µg/ml.
The numbers of small and large colonies and their analysis are presented in Tables 10 and 13. - Conclusions:
- The test item did not induce any statistically significant or dose related increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test. This study is considered to be scientifically justified for use as a key study under Regulation (EC) No. 1907/2006.
- Executive summary:
Introduction.
The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese EPA/METI/MHLW guidelines for testing of new chemical substances.
Methods.
Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-Hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at six dose levels using a 4‑Hour exposure group in the presence of metabolic activation (1% S9) and a 24-Hour exposure group in the absence of metabolic activation.
The dose range of test item was selected following the results of a preliminary toxicity test and for the first experiment was 23.56 to 1508 µg/ml in the absence and presence of metabolic activation. For the second experiment the dose range was 94.25 to 1508 µg/ml in the absence and presence of metabolic activation.
Results.
The maximum dose level used in the mutagenicity test was the maximum recommended dose (10mM) of 1508 µg/ml. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.
The test item did not induce any statistically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.
Conclusion.
The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.
Referenceopen allclose all
Table 1: Historical control data of negative and positive control values of the years 2012-2014 (n=97 studies)
Data obtained from plate incorporation and preincubation tests
Negative Reference Item
Strain |
TA98 |
|
TA100 |
|
TA102 |
|
TA1535 |
|
TA1537 |
|
S9 -mix |
- |
+ |
- |
+ |
- |
+ |
- |
+ |
- |
+ |
Mean |
29.9 |
31.4 |
144.0 |
143.7 |
275.3 |
279.3 |
18.6 |
18.1 |
6.5 |
6.6 |
SD |
6.1 |
6.3 |
20.5 |
20.2 |
16.0 |
16.6 |
4.5 |
4.5 |
2.1 |
2.3 |
Min |
12 |
18 |
100 |
101 |
224 |
245 |
10 |
8 |
2 |
0 |
Max |
49 |
50 |
191 |
189 |
319 |
319 |
31 |
42 |
11 |
18 |
Positive Reference Item
Strain |
TA98 |
|
TA100 |
|
TA102 |
|
TA1535 |
|
TA1537 |
|
S9 -mix |
- |
+ |
- |
+ |
- |
+ |
- |
+ |
- |
+ |
|
2-NF |
BP |
SA |
2-AA |
MMC |
BP |
SA |
2-AA |
9-AC |
BP |
Mean |
178.8 |
176.9 |
925.2 |
932.9 |
972.4 |
963.8 |
140.4 |
142.6 |
90.9 |
90.4 |
SD |
65.1 |
65.1 |
96.3 |
88.4 |
115.8 |
104.8 |
44.8 |
45.6 |
40.0 |
38.4 |
Min |
91 |
83 |
463 |
703 |
759 |
757 |
51 |
49 |
26 |
28 |
Max |
434 |
433 |
1209 |
1181 |
1637 |
1571 |
382 |
371 |
272 |
257 |
2-NF : 2-nitro-fluorene
BP: Benzo[a]pyrene
SA: Sodium azide
2-AA: 2-amino-anthracene
MMC: Mitomycin C
9-AC: 9-amino-acridine
Main test
Table 1: Plate incorporation test; without metabolic activation
Test substance µg/plate |
|
Number of reverted colonies |
||||
|
|
TA98 |
TA100 |
TA102 |
TA1535 |
TA1537 |
31.6 |
mean |
34.7 |
120.0 |
288.7 |
19.0. |
9.0 |
|
SD |
2.1 |
3.6 |
11.8 |
5.3 |
1.0 |
100 |
mean |
36.0 |
119.0 |
258.3 |
15.7 |
9.3 |
|
SD |
2.6 |
2.6 |
2.9 |
5.5 |
0.6 |
316 |
mean |
30.7 # |
123.0 # |
284.0 # |
16.3 # |
8.3 # |
|
SD |
8.7 |
6.1 |
2.6 |
1.2 |
1.2 |
500 |
mean |
24.0 # |
137.7 # |
264.7 # |
18.0 # |
6.0 # |
|
SD |
3.5 |
3.2 |
1.5 |
1.0 |
1.0 |
2000 |
mean |
21.0 # |
112.0 # |
287.7 # |
21.3 # |
6.0 # |
|
SD |
1.7 |
1.0 |
6.4 |
4.2 |
1.7 |
5000 |
mean |
10.7 # |
59.3 # |
156.7 # |
7.3 # |
1.7 # |
|
SD |
0.6 |
9.9 |
0.6 |
3.1 |
0.6 |
Negative reference item 100 μL/plate |
||||||
|
mean |
27.0 |
140.3 |
259.3 |
18.0 |
8.3 |
|
SD |
1.0 |
1.2 |
4.2 |
4.4 |
0.6 |
Positive reference item |
|
2-NF |
SA |
MMC |
SA |
9-AC |
Concentration μg/plate |
|
10 |
10 |
10 |
10 |
100 |
|
mean |
154.0 |
966.7 |
1198.0 |
136.3 |
114.7 |
|
SD |
11.3 |
2.5 |
25.4 |
10.7 |
4.0 |
2-NF : 2-nitro-fluorene
SA: Sodium azide
MMC: Mitomycin C
9-AC: 9-amino-acridine
# test item precipitation
SD standard deviation
mean (n = 3)
Table 2: Plate incorporation test; with metabolic activation
Test substance µg/plate |
|
Number of reverted colonies |
||||
|
|
TA98 |
TA100 |
TA102 |
TA1535 |
TA1537 |
31.6 |
mean |
24.0 |
121.7 |
270.3 |
15.7 |
5.7 |
|
SD |
2.6 |
15.4 |
3.8 |
1.2 |
1.2 |
100 |
mean |
24.3 |
119.0 |
268.0 |
18.7 |
6.0 |
|
SD |
4.2 |
10.4 |
6.1 |
6.4 |
0.0 |
316 |
mean |
25.3 # |
134.7 # |
279.7 # |
13.7 # |
7.3 # |
|
SD |
2.1 |
8.5 |
20.2 |
0.6 |
2.3 |
500 |
mean |
37.7 # |
113.7 # |
261.3 # |
14.7 # |
6.3 # |
|
SD |
0.6 |
0.6 |
11.6 |
2.5 |
2.5 |
2000 |
mean |
34.0 # |
122.7 # |
293.7 # |
13.3 # |
5.3 # |
|
SD |
1.0 |
1.2 |
4.2 |
0.6 |
1.2 |
5000 |
mean |
11.3 # |
70.7 # |
128.3 # |
7.3 # |
1.7 # |
|
SD |
0.6 |
3.2 |
0.6 |
1.2 |
0.6 |
Negative reference item 100 μL/plate |
||||||
|
mean |
27.7 |
119.0 |
274.3 |
14.0 |
6.7 |
|
SD |
0.6 |
3.0 |
18.8 |
1.0 |
1.5 |
Positive reference item |
|
BP |
2-AA |
BP |
2-AA |
BP |
Concentration μg/plate |
|
10 |
2 |
10 |
2 |
100 |
|
mean |
157.0 |
963.3 |
1161.0 |
135.3 |
104.7 |
|
SD |
8.2 |
6.0 |
2.0 |
15.6 |
17.2 |
BP: Benzo[a]pyrene
2-AA: 2-amino-anthracene
Table 3: Preincubation test; without metabolic activation
Test substance µg/plate |
|
Number of reverted colonies |
||||
|
|
TA98 |
TA100 |
TA102 |
TA1535 |
TA1537 |
31.6 |
mean |
23.0 |
146.0 |
264.7 |
20.3 |
8.0 |
|
SD |
0.0 |
4.4 |
1.2 |
0.6 |
1.7 |
100 |
mean |
27.7 |
144..3 |
286.3 |
16.7 |
5.0 |
|
SD |
1.5 |
7.0 |
1.5 |
5.1 |
1.0 |
316 |
mean |
30.3 # |
148.3 # |
272.7 # |
18.3 # |
7.3 # |
|
SD |
1.2 |
7.5 |
11.0 |
0.6 |
0.6 |
500 |
mean |
26.3 # |
134.0 # |
289.0 # |
19.0 # |
7.3 # |
|
SD |
4.6 |
10.0 |
5.3 |
3.6 |
0.6 |
2000 |
mean |
23.0 # |
122.3 # |
264.3 # |
13.3 # |
6.3 # |
|
SD |
2.6 |
2.1 |
22.2 |
2.3 |
1.5 |
5000 |
mean |
16.3 # |
82.3 # |
182.0 # |
8.3 # |
1.0 # |
|
SD |
3.1 |
4.0 |
25.0 |
0.6 |
0.0 |
Negative reference item 100 μL/plate |
||||||
|
mean |
26.3 |
146.3 |
288.3 |
19.0 |
5.0 |
|
SD |
5.9 |
2.9 |
14.4 |
7.8 |
0.0 |
Positive reference item |
|
2-NF |
SA |
MMC |
SA |
9-AC |
Concentration μg/plate |
|
10 |
10 |
10 |
10 |
100 |
|
mean |
144.3 |
916.3 |
887.3 |
110.7 |
52.0 |
|
SD |
2.1 |
10.4 |
4.7 |
15.0 |
1.7 |
2-NF : 2-nitro-fluorene
SA: Sodium azide
MMC: Mitomycin C
9-AC: 9-amino-acridine
Table 4: Preincubation test; with metabolic activation
Test substance µg/plate |
|
Number of reverted colonies |
||||
|
|
TA98 |
TA100 |
TA102 |
TA1535 |
TA1537 |
31.6 |
mean |
23.7 |
146.0 |
257.3 |
17.3 |
3.7 |
|
SD |
0.6 |
9.8 |
5.5 |
3.2 |
0.6 |
100 |
mean |
27.7 |
139.3 |
274.0 |
17.0 |
7.7 |
|
SD |
1.2 |
5.0 |
24.3 |
7.2 |
1.2 |
316 |
mean |
29.0 # |
147.3 # |
257.3 # |
16.3 # |
7.0 # |
|
SD |
2.0 |
3.1 |
3.5 |
4.0 |
2.6 |
500 |
mean |
27.3 # |
149.3 # |
275.0 # |
23.7 # |
7.0 # |
|
SD |
1.2 |
2.1 |
10.6 |
4.7 |
0.0 |
2000 |
mean |
30.0 # |
141.3 # |
275.3 # |
19.0 # |
8.3 # |
|
SD |
1.7 |
38.8 |
4.0 |
1.0 |
1.2 |
5000 |
mean |
17.0 # |
69.0 # |
191.0 # |
7.7 # |
1.0 # |
|
SD |
0.0 |
16.8 |
2.0 |
1.5 |
0.0 |
Negative reference item 100 μL/plate |
||||||
|
mean |
34.7 |
128.3 |
281.3 |
14.7 |
6.0 |
|
SD |
1.2 |
15.3 |
27.5 |
0.6 |
2.6 |
Positive reference item |
|
BP |
2-AA |
BP |
2-AA |
BP |
Concentration μg/plate |
|
10 |
2 |
10 |
2 |
10 |
|
mean |
165.3 |
910.7 |
906.7 |
116.3 |
56.3 |
|
SD |
7.2 |
5.5 |
7.6 |
11.5 |
15.1 |
BP: Benzo[a]pyrene
2-AA: 2-amino-anthracene
Table 1: 4-h exposure; without metabolic activation (S9 mix)
|
4-h exposure; without metabolic activation (S9 mix) |
|||
Concentration [μg/mL medium] |
CBPI |
RI [%] |
Number of binucleate cells scored |
Number of micronucleated cells per 1000 binucleate cells |
0.05 M HCl |
|
|
|
|
0 |
1.40 |
100 |
2000 |
6.5 |
Test item |
|
|
|
|
0.3162 |
1.39 |
98 |
2000 |
4.5 |
1.0 |
1.44 |
112 |
2000 |
7.0 |
3.162 |
1.44 |
112 |
2000 |
7.0 |
10.0 # |
1.46 |
115 |
2000 |
6.0 |
Mitomycin C |
|
|
|
|
0.2 |
1.35 |
87 |
2000 |
23.0 |
CBPI= Cytokinesis block proliferation index
RI= Cytokinesis block proliferation index
s. = significantly different from negative control (p < 0.05), CHI-SQUARE Test
# = test item precipitation
Table 2: 24-h exposure; without metabolic activation (S9 mix)
|
24-h exposure; without metabolic activation (S9 mix) |
|||
Concentration [μg/mL medium] |
CBPI |
RI [%] |
Number of binucleate cells scored |
Number of micronucleated cells per 1000 binucleate cells |
0.05 M HCl |
|
|
|
|
0 |
1.56 |
100 |
2000 |
6.5 |
Test item |
|
|
|
|
1.0 |
1.51 |
92 |
2000 |
5.0 |
3.162 |
1.47 |
84 |
2000 |
7.0 |
10.0 # |
1.42 |
75 |
2000 |
4.0 |
Colchicine |
|
|
|
|
0.02 |
1.52 |
94 |
2000 |
29.0s. |
Table 3: 4-h exposure; with metabolic activation (S9 mix)
|
4-h exposure; with metabolic activation (S9 mix) |
|||
Concentration [μg/mL medium] |
CBPI |
RI [%] |
Number of binucleate cells scored |
Number of micronucleated cells per 1000 binucleate cells |
0.05 M HCl |
|
|
|
|
0 |
1.52 |
100 |
2000 |
7.0 |
Test item |
|
|
|
|
0.3162 |
1.48 |
92 |
2000 |
7.0 |
1.0 |
1.51 |
97 |
2000 |
5.5 |
3.162 |
1.47 |
90 |
2000 |
5.5 |
10.0 # |
1.47 |
91 |
2000 |
6.0 |
Cyclophosphamide |
|
|
|
|
20 |
1.41 |
79 |
2000 |
18.5 s. |
Table 4: Historical Background data in vitro Micronucleus Test in cultured human peripheral lymphocytes
The micronucleus frequencies of the vehicle controls without and with metabolic activation for the last 13 studies (most recent background data of the years 2013 to 2015, not audited by the QAU-department) are given as follows:
|
Micronucleus frequency per 1000 cells |
|||
|
Without metabolic activation |
With metabolic activation |
||
|
Untreated control |
Vehicle control |
Untreated control |
Vehicle control |
mean |
6.6 |
6.3 |
6.4 |
6.1 |
SD |
2.9 |
3.1 |
2.6 |
4.2 |
range |
2.0 - 17 |
2.0 - 18 |
3.0 - 13 |
1 - 22 |
95% Confidence interval |
5.9 - 7.3 |
5.7 - 6.8 |
5.7 - 7.1 |
5.1 - 6.7 |
|
Mitomycin C Positive control |
Colchicine Positive control |
Cyclophosphamide Positive control |
|
mean |
46.9 |
25.9 |
44.0 |
|
SD |
35.0 |
9.5 |
37.7 |
|
range |
17 - 137 |
15 - 63 |
14 - 158 |
|
SD = Standard deviation
No further untreated negative controls (lacking solvent/vehicle) were used in this direct test, as the laboratory historical control data demonstrate that no genotoxic or other deleterious effects are induced by the chosen solvent at the concentrations used, as required by the guideline. Positive controls were always statistically significantly higher than controls in the corresponding studies.
Please see Attached ''Tables 1 -10''
Due to the nature and quantitiy of tables it was not possible to insert them into this section.
Table 1 Summary of Results
Table 2 Cell and 96-Well Plate Counts: Experiment 1 (-S9) 4-Hour Exposure
Table 3 Statistical Analysis: Experiment 1 (-S9) 4-Hour Exposure
Table 4 Large and Small Colonies Analysis: Experiment 1 (-S9) 4-Hour Exposure
Table 5 Cell and 96-Well Plate Counts: Experiment 1 (+S9) 4-Hour Exposure
Table 6 Statistical Analysis: Experiment 1 (+S9) 4-Hour Exposure
Table 7 Large and Small Colonies Analysis: Experiment 1 (+S9) 4-Hour Exposure
Table 8 Cell and 96-Well Plate Counts: Experiment 2 (-S9) 24-Hour Exposure
Table 9 Statistical Analysis: Experiment 2 (-S9) 24-Hour Exposure
Table 10 Large and Small Colonies Analysis: Experiment 2 (-S9) 24-Hour Exposure
Table 11 Cell and 96-Well Plate Counts: Experiment 2 (+S9) 4-Hour Exposure
Table 12 Statistical Analysis: Experiment 2 (+S9) 4-Hour Exposure
Table 13 Large and Small Colonies Analysis: Experiment 2 (+S9) 4-Hour Exposure
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Bacterial mutagenicity
In the GLP and OECD 471 compliant bacterial reverse mutation study (Ames test) the test item was tested for mutagenicity in 5 Salmonella typhimurium strains (LPT, 2015).
Following strains were used TA98, TA100, TA102, TA1535 and TA1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test.
In the Preliminary test ranging from 0.316 to 5000 μg/plate the top concentration for the main study was determined to be 5000 μg/plate, where no signs of cytotoxicity were noted.
In the main study the test item was suspended in 0.05 M HCl solution for concentrations of 316, 500, 2000 or 5000 μg per plate. For concentrations lower than 100 μg/plate, the test item was completely dissolved. The vehicle 0.05 M HCl solution served as the negative control.
Test item precipitation was noted in both experiments, each carried out without and with metabolic activation, at concentrations of 316 μg/plate and higher in all test strains. In addition, cytotoxicity (reduction of the number of revertants by more than 50%) was noted in the following experiments at the top concentration of 5000 μg/plate:
Plate incorporation test:
without S9: in test strains TA98, TA100, TA1535 and TA1537;
with S9: in test strains TA98, TA102 and TA1537;
Preincubation:
without S9 in test strains: TA1535 and TA1537
with S9 in test strains: TA98 and TA1537
No increase in revertant colony numbers as compared with control counts was observed for the test item, tested up to concentration of 5000 μg/plate in any of the 5 test strains in two independent experiments without and with metabolic activation. The positive control items showed a significant increase in the number of colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.
The study (Brusick, 1975) to assess the genetic toxicity to bacteria was designed comparable to OECD 471, however, the study was lacking as it only considered 3 types of S. typhimurium and did not include strain designed to detect cross-linking mutagens, the positive controls differ from those recommended in the guidelen and only one dose was investigated and is therefore only considered to be a reliability 3 study.
Cytogencity
In a GLP and OECD 487 compliant study the test item was tested in an in vitro micronucleus test using human peripheral lymphocytes (LPT, 2016).
The test was carried out employing 2 exposure times without S9 mix: 4 and 24 hours, and 1 exposure time with S9 mix: 4 hours. The harvesting time was 20 hours after the end of exposure. The cytokinesis-block technique was applied. The test item was suspended in 0.05 M HCl solution for a concentration of 10 μg per mL medium. For concentrations lower than 10 μg/mL medium, the test item was completely dissolved. The vehicle 0.05 M HCl solution served as the negative control. In a preliminary experiment 10.0 μg/mL were employed as the top concentration for the genotoxicity tests without and with metabolic activation.
In the main study test item precipitation was noted in the experiments without and with metabolic activation at the top concentration of 10.0 μg test item/mL medium. No signs of cytotoxicity were noted. Mitomycin C (at 0.2 μg/mL) and Colchicine (at 0.02 μg/mL) were employed as positive controls in the absence and Cyclophosphamide (at 20 μg/mL) in the presence of metabolic activation.
The cells were treated with the test item concentrations of 0.3162, 1.0, 3.162 or 10.0 μg/mL. There was no dose-related increase in micronuclei up to the top concentration of 10.0 μg/mL neither in the tests without metabolic activation (4- and 24-hour exposure) nor in the test with metabolic activation (4-hour exposure). The frequency of micronucleated cells was within the historical control range of the untreated and vehicle controls.
The test item revealed no indications of chromosomal damage in the in vitro micronucleus test in the absence and in the presence of metabolic activation, under the conducted test conditions.
Mammalian mutagenicity
A publication is available where the mutagenic activity of elemental and salt forms of iron (Fe), including ferric phosphate, were evaluated for mutagenicity in L5178Y mouse lymphoma cells (Dunkel, 1999). The study was performed similar to OECD 490. However, no differentiation between small and large colonies were performed. With ferric phosphate, there was an increase in mutant frequency only with metabolic acitvation. A mutant frequency (MF) of 117 was observed at the highest concentration and a relative total growth rate (RTG) of 16.5% compared to the solvent control where a MF of 43 was observed. The next lower concentration revealed a MF of 83 at a RTG of 60%. All other concentrations did not show an increase in MF. According to the test guideline the global evaluation factor (GEF) should be above 90 x 10E-6 in order to conclude for a positive reaction and care should be taken when interpreting positive results only found between 20 and 10% RTG. For this study only the highest concentration showed a clear postive result where the MF exceeds the GEF however in this case the positive result is found between 20 and 10% RTG and thus has to be considered with care. As the results are not clear a mouse lymphoma assay with the test substance was performed in order to find a final conclusion.
The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The study was performed according to OECD 476 and GLP compliant (Harlan, 2011).
Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-Hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at six dose levels using a 4‑Hour exposure group in the presence of metabolic activation (1% S9) and a 24-Hour exposure group in the absence of metabolic activation.
The dose range of test item was selected following the results of a preliminary toxicity test and for the first experiment was 23.56 to 1508 µg/mL in the absence and presence of metabolic activation. For the second experiment the dose range was 94.25 to 1508 µg/mL in the absence and presence of metabolic activation.
The maximum dose level used in the mutagenicity test was the maximum recommended dose (10 mM) of 1508 µg/mL. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.
In this study the test item did not induce any statistically significant or dose-related increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.
Conclusion
The test item was determined to be not mutagenic in the bacterial reserves mutation test, in vitro mammalian cell micronucleus and in the in vitro mammalian cell gene mutation test.
Justification for classification or non-classification
The available data on genetic toxicity do not meet the criteria for classification according to the CLP Regulation (EC) No. 1272/2008 and are therefore conclusive but not sufficient for classification.
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.
