Registration Dossier
Registration Dossier
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EC number: 202-532-0 | CAS number: 96-76-4
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The substance was negative in a reliable Ames test (bacterial reverse mutation assay) employing Salmonella typhimurium strains TA1535, TA98, TA97a, TA102 and TA100, both in the presence and absence of a metabolic activation system (rat liver S9) at concentrations of up to 1500 µg/plate.
The substance gave ambigious results in the chromosome aberration test employing cultured Chinese hamster lung fibroblasts, in the presence or absence of a metabolic activation system (rat liver S9) at concentrations of up to 10 µg/L (without S9) and 6 µg/L (with S9).
A read-across substance (2,6 -bis(1,1 -dimethyethyl)-phenol) did not induce gene mutations at the HGPRT locus in Chinese hamster V79 cells at concentrations of up to 8 µg/mL (without S9) and 50 µg/mL (with S9).
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- Batch-no.: 1437
Purity: min. 99% - Species / strain / cell type:
- S. typhimurium, other: TA97a
- Species / strain / cell type:
- S. typhimurium TA 98
- Species / strain / cell type:
- S. typhimurium TA 100
- Species / strain / cell type:
- S. typhimurium TA 102
- Species / strain / cell type:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 enzymes from the livers male Sprague-Dawley rats, treated with 500 mg Aroclor 1254/kg bw
- Test concentrations with justification for top dose:
- Toxicity test: 5000 / 1500 / 500 / 150 / 50 µg/plate
First experiment: 1500 / 500 / 150 / 50 / 15 µg/plate
second experiment: 1500 / 750 / 375 / 188 / 94 / 47 / 23 / 12 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: In a preliminary test, the solubility of the test item was determined in demineralised water and DMSO. The test item was only soluble in a concentration of 50 g/L in DMSO. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-Nitro-1,2-phenylene, 20 µg/plate with strains TA97a, TA98 and TA102; Sodium Azide, 1 µg/plate with strains TA100 and TA1535
- Remarks:
- without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Amino-anthracene, 1 µg/plate with strains TA97a, TA100, TA102 and TA1535; Benzo-a-pyrene, 20 µg/plyte with strain TA98
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) in the first experiment; preincubation in the second experiment
DURATION
- Preincubation period: 20 min (only in the second experiment)
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY:
- Method: In the pre-experiment: determination of titre (the test item was considered non-toxic, if the quotient titre/toxicity is below 2), in the main experiments: evaluation of background lawn, reduction in number of revertants in comparison to negativ/solvents control
OTHER EXAMINATIONS:
- Visual counting of mutant colonies, a spreadsheet software (Microsoft Excel) was used to calculate mean values and standard deviations.
- Quality control of bacterial strains: genotype confirmation for each batch of bacteria before stock culture preparation: all bacterial strains were tested for histidine requirement, ampicillin resistence, crystal violet sensitivity, UV sensitivity and spontaneous revertants, furthermore the following examinations were performed: determination of titre, toxicity control, sterility control and positive control - Evaluation criteria:
- The colonies were counted visually, the numbers were recorded. A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control. The increase factor f(I) of revertant induction (mean revertants divided by mean spontaneous revertants) and the absolute number of revertants (revertants less mean spontaneous revertants) were also calculated.
A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate in at least one strain exceeding an increase factor of 2 (in tester strains TA 97a, TA98, TA100 and TA102) and an increase factor of 3 (in tester strain TA1535) as compared to the reversion rate of the solvent control can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity. - Statistics:
- not performed
- Species / strain:
- S. typhimurium, other: TA 97a
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- in the preincubation experiment at 1500 and 750 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- in the preincubation experiment at 1500 and 750 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- in the preincubation experiment at 1500 and 750 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- in the preincubation experiment at 1500 and 750 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- in the preincubation experiment at 1500 and 750 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: not soluble in water
- Precipitation: Precipitated/undissolved test item was not observed at any of the concentrations tested.
- Other confounding effects: nothing mentioned
RANGE-FINDING/SCREENING STUDIES: A pre-experiment for toxicity was performed according to the plate incorporation method. The toxicity of the following concentrations were tested: 5000 / 1500 / 500 / 150 / 50 µg/plate. Toxicity was observed in all tester strains only in the highest concentration.
COMPARISON WITH HISTORICAL CONTROL DATA: Nearly all determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory, differences were only marginal and no critical impact on the outcome of the study was expected. All positive control showed mutagenic effects with and without metabolic activation. - Conclusions:
- The test item did not show mutagenic effects towards Salmonella typhimurium, strains TA97a, TA98, TA100, TA102 and TA1535. Therefore, no concentration-effect relationship could be determined. The test item 2,4-di-tert-butylphenol is considered as "not mutagenic under the conditions of the test":
- Executive summary:
Two valid experiments were performed following OECD 471 and EU B.13/14 under the conditions of GLP.
First Experiment: On the base of a pre-test for toxicity, five concentrations of the test item, dissolved in DMSO (ranging from 15 to 1500 μg/plate) were used. Five genetically changed strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 (genetically manipulated) and TA1535) were exposed to the test item both in the presence and in the absence of a metabolic activation system (S9-mix, rat liver S9-mix induced by Aroclor 1254) for 48 hours, using the plate incorporation method. None of the concentrations caused a significant increase in the number of revertant colonies in the tested strains. The test item did not show any mutagenic effects in the first experiment. The test item showed no precipitates on the plates in all tested concentrations. No signs of toxicity towards the bacteria could be observed. The sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.
Second Experiment: To verify the results of the first experiment, a second experiment was performed, using eight concentrations of the test item (ranging from 12 to 1500 μg/plate) and a modification in study performance (pre-incubation method). The test item did not show mutagenic effects in the second experiment, either. The test item showed no precipitates on the plates in all tested concentrations. Signs of toxicity towards all bacteria strains could be observed in the two highest concentrations (1500 and 750 μg/plate). The sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.
Under the conditions of the test, the test item did not show mutagenic effects towards Salmonella typhimurium, strains TA97a, TA98, TA100, TA102 and TA1535. Therefore, no concentration-effect relationship could be determined. The test item 2,4-di-tert-butylphenol is considered as “not mutagenic under the conditions of the test”.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1998-05-19 to 1998-08-13
- 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)
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- Purity: 99.72%
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM (lot #E05828-015), purchased from PAA Laboratories GmbH, Germany
- Properly maintained: yes, stock culture maintained under liquid nitrogen
- Periodically checked for Mycoplasma contamination: not mentioned
- Periodically checked for karyotype stability: not mentioned
- Periodically "cleansed" against high spontaneous background: not applicable - Additional strain / cell type characteristics:
- other: cell cycle length approx. 16 hours
- Metabolic activation:
- with and without
- Metabolic activation system:
- liver microsomal fraction (S9 mix), Phenobarbital/ß-Naphthoflavone induced
- Test concentrations with justification for top dose:
- 1 / 2 / 4 / 6 / 10 / 20 / 40 / 60 / 80 / 100 µg/ml in the first test
1 / 4 / 5 / 6 / 10 µg/ml in the second test - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone, 1% (v/v)
- Justification for choice of solvent/vehicle: The test substance was soluble in acetone. In MEM medium containing 1% acetone, the solubility limit of the test subtance was determined to be 100 µg/ml (homogeneous emulsion). - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- MEM medium containing 1% acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- without metabolic activation Migrated to IUCLID6: 0.02 and 0.03 µg/ml
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- with metabolic activation Migrated to IUCLID6: 2 and 3 µg/ml
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 24 hours
- Exposure duration: 20 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours
SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.2 µg/ml final conc.)
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: 2 per experimental point
NUMBER OF CELLS EVALUATED: 2000 per experimental point for determination of mitotic index; 200 metaphases per experimental point for analysis of chromosomal aberrations
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: Osmolality and pH of the highest test substance concentration
OTHER: none - Evaluation criteria:
- The test is considered valid if the following criteria were met:
1. The number of cells with chromosomal aberrations (excl. gaps) in the negative control group falls within the normal range (< 5 %). The percentage of polyploid and endoreduplicated cells should be < 10 %.
2. Approx. 200 cells per treatment group (approx. 100 for each positive control) are analysable for chromosomal aberrations.
3. Treatment with positive controls (MMC and CP) in at least one concentration tested leads to clear increases in the frequency of cells with structurally altered chromosomes (>> 5 % excl. gaps).
The test chemical is to be considered clastogenic in this assay if
1. it induces chromosomal aberrations (excl. gaps) in a statistically significant manner in one or more concentrations
2. the induced proportion of aberrant cells at such test substance concentrations exceeds the normal range of the test system (i.e. >> 5 %)
3. positive results can be verified in an independent experiment.
The possible influence of pH, S9 mix or osmolality on the occurrence of chromosomal aberrations will also be considered. - Statistics:
- The proportion of cells that was treated with the test substance and harboured structural aberrations (excl. gaps) was compared with the corresponding proportion of the negative controls in the Chi-square test. Probability values of p < 0.05 were accepted as statistically significant
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: not applicable
- Water solubility: unsoluble
- Precipitation: no precipitation up to 100 µg/ml
- Other confounding effects: none
RANGE-FINDING/SCREENING STUDIES: 10 different concentrations in the range of 1 - 100 µg/ml 2,4-di-tert Butylphenol were tested in the presence and absence of exogenous metabolic activation. On the basis of the determination of the mitotic index, the following concentrations were scored for chromosomal aberration induction: 0 / 1 / 6 / 10 µg/ml without metabolic activation in the first and second test; 0 / 1 / 4 / 6 µg/ml with metabolic activation in the first test, 0 / 1 / 4 / 5 / 6 µg/ml with metabolic activation in the second test.
COMPARISON WITH HISTORICAL CONTROL DATA: yes, the negative controls revealed chromosomal aberration frequencies which were consistent with spontaneous aberration frequencies for the V79 cells of the testing laboratory
ADDITIONAL INFORMATION ON CYTOTOXICITY: The experiments revealed a systematic influence of the test substance resulting in a reduction of the mitotic index (see tables below).
- With metabolic activation: First occurrence of cytotoxicity at 5 µg/ml in test #1, from 10 to 100 µg/ml no cells were on the slides. In test #2 cytotoxic effects were seen even at a concentration of 4 µg/ml, above 5 µg/ml test substance no cells were on the slides.
- Without metabolic activation: First occurrence of cytotoxicity at 6 µg/ml, in the range of 20 to 100 µg/ml no cells could be found on the slides.
GENOTOXIC EFFECTS:
- With metabolic activation: Treatment with the top concentration of 6 µg/ml 2,4-Di-tert-butylphenol resulted in an effect of very high statistical significance (P < 0.001) in test #1. The concentration of 4 µg/ml did not result in statistically or biologically significant increases in the frequency of cells with chromosome aberrations excluding gaps. In order to establish a dose response relationship, in the repeat experiment (test #2 with metabolic activation) an additional fourth test compound concentration of 5 µg/ml was introduced. Treatment with 5 µg/ml resulted in an effect of very high statistically significance (P < 0.001), 4 µg/ml 2,4-Di-tert-butylphenol showed no statistically significant effects in the aberration frequency in relation to the negative control. The determination of the mitotic index of test #2 showed a high cytotoxic effect in the concentration of 6 µg/ml 2,4-Di-tert-butylphenol, a scoring of aberrations was not possible. (See tables below).
- Without metabolic activation: In both experiments (#1 and #2) treatment with 2,4-Di-tert-butylphenol did not result in statistically or biologically significant increases in the frequency of cells with chromosome aberrations excluding gaps. (See tables below).
Due to the positive response of the test substance at the 20 hours sampling time, scoring of slides of the additional 28 hours sampling time was considered to be obsolete. - Conclusions:
- It is concluded, that in both experiments with metabolic activation, treatment with 2,4-di-tert-Butylphenol resulted in chromosomal aberration frequencies being significantly higher (biologically and statistically) than negative control values in the highest concentrations. A dose response relationship could not be established statistically in the frequency of cells with chromosomal aberrations excluding gaps. A connection between the chromosomal aberrations and the high cytotoxicity of the test substance can not be excluded.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- 1. HYPOTHESIS FOR THE ANALOGUE APPROACH
2,4-DTBP which is structurally similar to 2,6-DTBP - Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Conclusions:
- In conclusion can be stated that during the described mutagenicity test and under the experimental conditions reported the test article did not induce point mutations at the HGPRT locus in V79 cells.
- Executive summary:
The study was performed to investigate the potential of 2,6 -bis(1,1 -dimethyethyl)-phenol to induce gene mutations at the HGPRT locus in V79 cells of the Chinese hamster in vitro.
The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation.
The test article was tested with the following concentrations:
Experiment I and II:
Without S9 mix: 0.3, 1 -0, 2.0, 4.0, 6.0*, and 8.0 µg/ml
With S9 mix: 3.0, 10.0, 20.0*, 30.0, 40.0, 50.0 µg/ml
* culture was not continued in experiment I and II
According to the pre-experiment for toxicity the concentration ranges were selected to yield concentration related toxic effects. In the first experiment the highest concentration produced a low level of survival and the survival at the lowest concentration was approximately in the range of the negative control.
At the highest investigated concentration no relevant increase in mutant colony numbers was obtained in two independent experiments.
Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies.
In conclusion it can be stated that during the described mutagenecity test and under the experimental conditions reported the test article did not induce point mutations at the HGPRT locus in V79 cells.
Referenceopen allclose all
Table #1: First Mutation Assay (Direct Plate Incorporation Method)
| TA 97a | TA 98 | TA 100 | ||||||||||
| - S9 mix | + S9 mix | - S9 mix | + S9 mix | - S9 mix | + S9 mix | |||||||
| Dose level[µg/plate] | Mean revertants per plate ± SD | f (I) | Mean revertants per plate ± SD | f (I) | Mean revertants per plate ± SD | f (I) | Mean revertantsper plate± SD | f (I) | Mean revertants per plate ± SD | f (I) | Mean revertants per plate ± SD | f (I) |
| H2O | 120 ± 2.3 | - | 1115 ± 4.5 | - | 19 ± 6.1 | - | 17 ± 4.0 | - | 91 ± 1.0 | - | 123 ± 6.4 | - |
| DMSO | 118 ± 4.2 | - | 116 ± 3.6 | - | 20 ± 2.6 | - | 22 ± 2.1 | - | 97 ± 9.5 | - | 114 ± 11.9 | - |
| 1500 | 133 ± 22.0 | 1.13 | 115 ± 4.4 | 0.99 | 12 ± 2.0 | 0.60 | 13 ± 2.3 | 0.59 | 113 ± 5.2 | 1.16 | 115 ± 4.0 | 1.01 |
| 500 | 116 ± 4.2 | 0.98 | 115 ± 4.7 | 0.99 | 13 ± 0.6 | 0.65 | 14 ± 1.7 | 0.64 | 117 ± 1.7 | 1.21 | 120 ± 5.5 | 1.05 |
| 150 | 112 ± 1.7 | 0.95 | 129 ± 6.1 | 1.11 | 10 ± 3.5 | 0.50 | 15 ± 3.8 | 0.68 | 106 ± 14.6 | 1.09 | 100 ± 11.1 | 0.88 |
| 50 | 123 ± 15.4 | 1.04 | 113 ± 7.6 | 0.97 | 13 ± 1.2 | 0.65 | 18 ± 0.6 | 0.82 | 129 ± 9.5 | 1.33 | 115 ± 18.2 | 1.01 |
| 15 | 118 ± 4.6 | 1.00 | 115 ± 5.6 | 0.99 | 17 ± 2.5 | 0.85 | 20 ± 4.2 | 0.91 | 105 ± 11.4 | 1.08 | 94 ± 8.4 | 0.81 |
| Positive controls | 497 ± 33.3 | 4.21 | 485 ± 22.0 | 4.18 | 493 ± 44.1 | 24.7 | 102 ± 10.3 | 4.64 | 731 ± 41.6 | 8.03 | 661 ± 31.1 | 5.80 |
f (I) = increase factor of revertant induction (mean revertants divided by mean spontaneous revertants)
Table #1 (continued): First Mutation Assay (Direct Plate Incorporation Method)
| TA 102 | TA 1535 | |||||||
| - S9 mix | + S9 mix | - S9 mix | + S9 mix | |||||
| Dose level [µg/plate] | Mean revertants per plate ± SD | f (I) | Mean revertants per plate ± SD | f (I) | Mean revertants per plate ± SD | f (I) | Mean revertants per plate ± SD | f (I) |
| H2O | 202 ± 8.7 | - | 199 ± 20.8 | - | 21 ± 1.0 | - | 22 ± 3.1 | - |
| DMSO | 198 ± 15.6 | - | 237 ± 5.0 | - | 20 ± 2.6 | - | 17 ± 4.0 | - |
| 1500 | 169 ± 12.2 | 0.85 | 229 ± 19.7 | 0.97 | 13 ± 3.8 | 0.65 | 13 ± 5.5 | 0.73 |
| 500 | 197 ± 10.1 | 0.99 | 219 ± 23.2 | 0.92 | 14 ± 4.4 | 0.70 | 11 ± 2.1 | 0.65 |
| 150 | 204 ± 17.4 | 1.03 | 219 ± 11.0 | 0.92 | 10 ± 0.6 | 0.50 | 13 ± 2.3 | 0.76 |
| 50 | 188 ± 33.6 | 0.95 | 224 ± 28.3 | 0.95 | 15 ± 3.8 | 0.75 | 12 ± 2.5 | 0.71 |
| 15 | 248 ± 45.4 | 1.25 | 208 ± 43.3 | 0.88 | 12± 2.5 | 0.60 | 14 ± 2.1 | 0.82 |
| Positiv controls | 779 ± 209.8 | 3.93 | 955 ± 90.0 | 4.03 | 133 ± 16.3 | 6.33 | 112 ± 14.0 | 6.59 |
f (I) = increase factor of revertant induction (mean revertants divided by mean spontaneous revertants)
Table #2: Second Mutation Assay (Pre-incubation Method)
| TA 97a | TA 98 | TA 100 | ||||||||||
| - S9 mix | + S9 mix | - S9 mix | + S9 mix | - S9 mix | + S9 mix | |||||||
| Dose level [µg/plate] | Mean revertants per plate ± SD | f (I) | Mean revertants per plate ± SD | f (I) | Mean revertants per plate ± SD | f (I) | Mean revertants per plate ± SD | f (I) | Mean revertants per plate ± SD | f (I) | Mean revertants per plate ± SD | f (I) |
| H2O | 129 ± 8.5 | - | 124 ± 16.9 | - | 14 ± 1.5 | - | 13 ± 0.0 | - | 135 ± 6.1 | - | 133 ± 13.1 | - |
| DMSO | 109 ± 7.8 | - | 109 ± 6.5 | - | 12 ± 2.6 | - | 10 ± 0.0 | - | 132 ± 5.9 | - | 139 ± 8.1 | - |
| 1500 | 0 ± 0.0 | 0 | 0 ± 0.0 | 0 | 0 ± 0.0 | 0 | 0 ± 0.0 | 0 | 0 ± 0.0 | 0 | 0 ± 0.0 | 0 |
| 750 | 10 ± 1.0 | 0.09 | 12 ± 6.7 | 0.11 | 1 ± 1.7 | 0.08 | 7 ± 4.9 | 0.70 | 12 ± 4.2 | 0.09 | 9 ± 5.2 | 0.06 |
| 375 | 129 ± 22.4 | 1.18 | 134 ± 13.3 | 1.23 | 10 ± 2.5 | 0.83 | 10 ± 0.6 | 1.00 | 40 ± 2.6 | 0.30 | 31 ± 14.6 | 0.22 |
| 188 | 117 ± 10.7 | 1.07 | 135 ± 8.7 | 1.24 | 13 ± 1.0 | 1.08 | 12 ± 1.2 | 1.20 | 110 ± 5.1 | 0.83 | 108 ± 11.1 | 0.78 |
| 94 | 107 ± 6.1 | 0.98 | 110 ± 1.0 | 1.01 | 11 ± 2.0 | 0.92 | 12 ± 2.1 | 1.20 | 122± 16.3 | 0.92 | 111 ± 5.5 | 0.80 |
| 47 | 105 ± 5.5 | 0.96 | 104 ± 5.1 | 0.95 | 13 ± 3.5 | 1.08 | 11 ± 1.7 | 1.10 | 117 ± 1.2 | 0.89 | 115 ± 10.1 | 0.83 |
| 23 | 112 ± 3.1 | 1.03 | 134 ± 12.5 | 1.23 | 15 ± 1.2 | 1.25 | 15 ± 3.1 | 1.50 | 110 ± 12.6 | 0.83 | 119 ± 23.6 | 0.86 |
| 12 | 109 ± 2.1 | 1.00 | 138 ± 12.9 | 1.27 | 10 ± 0.6 | 0.83 | 11 ± 1.2 | 1.10 | 130 ± 13.6 | 0.98 | 96 ± 14.2 | 0.69 |
| Positive controls | 499 ± 44.1 | 4.58 | 456 ± 73.0 | 4.18 | 109 ± 1.2 | 9.08 | 99 ± 10.1 | 9.90 | 555 ± 36.1 | 4.11 | 717 ± 184.0 | 5.16 |
f (I) = increase factor of revertant induction (mean revertants divided by mean spontaneous revertants)
Table #3 (continued): Second Mutation Assay (Pre-incubation Method)
| TA 102 | TA 1535 | |||||||
| - S9 mix | + S9 mix | - S9 mix | + S9 mix | |||||
| Dose level [µg/plate] | Mean revertants per plate ± SD | f (I) | Mean revertants per plate ± SD | f (I) | Mean revertants per plate ± SD | f (I) | Mean revertants per plate ± SD | f (I) |
| H2O | 365 ± 17.0 | - | 370 ± 23.6 | - | 14 ± 4.0 | - | 10 ± 0.6 | - |
| DMSO | 351 ± 68.9 | - | 397 ± 19.7 | - | 14 ± 3.1 | - | 14 ± 3.2 | - |
| 1500 | 114 ± 17.5 | 0.32 | 7 ± 5.5 | 0.02 | 0 ± 0.0 | 0 | 3 ± 1.5 | 0.21 |
| 750 | 107 ± 14.6 | 0.30 | 86 ± 11.0 | 0.22 | 4 ± 2.3 | 0.29 | 6 ± 0.0 | 0.43 |
| 375 | 210 ± 20.0 | 0.60 | 210 ± 47.6 | 0.53 | 8 ± 1.5 | 0.57 | 10 ± 0.6 | 0.71 |
| 188 | 267 ± 56.6 | 0.76 | 361± 27.2 | 0.91 | 15 ± 3.1 | 1.07 | 16 ± 2.6 | 1.14 |
| 94 | 313 ± 31.9 | 0.89 | 363 ± 9.2 | 0.91 | 12 ± 3.1 | 0.86 | 12 ± 2.1 | 0.86 |
| 47 | 361 ± 64.0 | 1.03 | 265 ± 28.4 | 0.67 | 16 ± 0.6 | 1.14 | 15 ± 3.1 | 1.07 |
23 |
275 ± 41.1 |
0.78 |
318 ± 56.0 |
0.80 |
11 ± 3.2 |
0.79 |
12 ± 2.6 |
0.86 |
| 12 | 421 ± 51.6 | 1.20 | 372 ± 65.5 | 0.94 | 17 ± 8.7 | 1.21 | 12 ± 4.0 | 0.86 |
Positive controls |
1076 ± 133.1 |
3.07 |
1071 ± 63.5 |
2.70 |
165 ± 39.3 |
11.8 |
134 ± 6.7 |
9.57 |
f (I) = increase factor of revertant induction (mean revertants divided by mean spontaneous revertants)
Influence on the mitotic index:
| test #1 | |||||
| without S9 mix | with S9 mix | ||||
| concentration[µg/ml] | absoluteMitotic index[%] | relativeMitotic index[%] | concentration[µg/ml] | absoluteMitotic index[%] | relativeMitotic index[%] |
| Neg. control | 6.3 | 100 | Neg. control | 5.6 | 100 |
| 1 | 5.6 | 88.9 | 1 | 4.7 | 84.7 |
| 6 | 5.1 | 81.0 | 4 | 6.7 | 120.7 |
| 10 | 4.4 | 69.0 | 6 | 2.5 | 44.1* |
| test #2 | |||||
| without S9 mix | with S9 mix | ||||
| concentration[µg/ml] | absoluteMitotic index[%] | relativeMitotic index[%] | concentration[µg/ml] | absoluteMitotic index[%] | relativeMitotic index[%] |
| Neg. control | 14.7 | 100 | Neg. control | 6.3 | 100 |
| 1 | 11.6 | 78.8 | 1 | 7.5 | 120.0 |
| 6 | 6.3 | 43.0* | 4 | 2.5 | 40.0* |
| 10 | 4.6 | 31.4* | 5 | 1.4 | 22.4* |
* relative mitotic indices lower than 50%
Genotoxic effects:
test #1
| concentration[µg/ml] | S9 mix | Total # of mitotic cells scored | Proportion of cells with aberrations excl. gaps[%] | significance |
| Neg. control | - | 200 | 0.0 | - |
| 1 | - | 200 | 0.0 | ns |
| 6 | - | 200 | 1.5 | ns |
| 10 | - | 400 | 1.8 | ns |
| Pos. controlMMC 0.03 | - | 183 | 17.5 | s |
| Neg. control | + | 200 | 1.0 | - |
| 1 | + | 200 | 0.5 | ns |
| 4 | + | 200 | 1.0 | n |
| 6 | + | 200 | 9.5 | *** |
| Pos. control CP 3 | + | 200 | 9.5 | s |
| Pos. control CP 4 | + | 200 | 18.0 | s |
test #2
| concentration[µg/ml] | S9 mix | Total # of mitotic cells scored | Proportion of cells with aberrations excl. gaps[%] | significance |
| Neg. control | - | 200 | 0.0 | - |
| 1 | - | 200 | 0.5 | ns |
| 6 | - | 201 | 2.5 | ns |
| 10 | - | 200 | 1.0 | ns |
| Pos. controlMMC 0.03 | - | 200 | 24.5 | s |
| Neg. control | + | 200 | 0.5 | - |
| 1 | + | 200 | 0.0 | ns |
| 4 | + | 200 | 3.5 | ns |
| 5 | + | 228 | 9.2 | *** |
| Pos. control CP 3 | + | 200 | 8.0 | s |
| Pos. control CP 4 | + | 200 | 12.0 | s |
ns = not significant s = significant, no statistical evaluation *** = statistically significant (P<0.001)
Pre-experiment for toxicity
(the results were obtained from CCR Project 243628, Chromosome aberration)
Plating Efficiency Assay without metabolic activation, 495 single cells were seeded into each flask
Conc. Per ml |
Colonies counted |
mean |
PE% relative |
|
Flask I |
Flask II |
|||
Negative control |
301 |
248 |
274.5 |
|
Solvent control (DMSO) |
280 |
271 |
275.5 |
100.0 |
0.1µg |
266 |
291 |
278.5 |
101.1 |
1.0µg |
221 |
259 |
240.0 |
87.1 |
3.0µg |
288 |
300 |
294.0 |
106.7 |
6.0µg |
2 |
9 |
5.5 |
2.0 |
10.0µg |
0 |
0 |
0.0 |
0.0 |
30.0µg |
0 |
0 |
0.0 |
0.0 |
50.0µg |
0 |
0 |
0.0 |
0.0 |
100µg |
0 |
0 |
0.0 |
0.0 |
Plating Efficiency Assay with metabolic activation, 495 single cells were seeded into each flask
onc. Per ml |
Colonies counted |
mean |
PE% relative |
|
Flask I |
Flask II |
|||
Negative control |
309 |
309 |
309.0 |
|
Solvent control (DMSO) |
299 |
271 |
285.0 |
100.0 |
0.1µg |
321 |
287 |
304.0 |
106.7 |
1.0µg |
313 |
281 |
297.0 |
104.2 |
3.0µg |
282 |
302 |
292.0 |
102.5 |
6.0µg |
301 |
299 |
300.0 |
105.3 |
10.0µg |
256 |
284 |
270.0 |
94.7 |
30.0µg |
320 |
279 |
299.5 |
105.1 |
50.0µg |
0 |
0 |
0.0 |
0.0 |
100µg |
0 |
0 |
0.0 |
0.0 |
The test article was tested in experiment I (without S9 mix) and II (with S9 mix).
In the first experiment the plating efficiency was reduced at the highest concentration but in the second experiment no decrease of the plating efficiency was observed up to the highest concentration (see tables I and IV), PE% relative).
Table I: toxicity data, experiment I
Column |
Conc. Per ml |
S9 mix |
Number of cells per flask* |
PE%** absolute |
PE%*** relative |
|||
seeded |
found |
mean |
||||||
I/II |
I |
II |
|
|||||
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
|
Negative control |
0.00µg |
- |
497 |
298 |
299 |
298.5 |
60.1 |
100.0 |
Solvent control DMSO |
0.00µg |
- |
497 |
260 |
260 |
260.0 |
52.3 |
100.0 |
Positive control EMS |
0.60µg |
- |
497 |
197 |
160 |
178.5 |
35.9 |
59.8 |
Test article |
0.30µg |
- |
497 |
266 |
257 |
261.5 |
52.6 |
100.6 |
Test article |
1.00µg |
- |
497 |
359 |
232 |
245.5 |
49.4 |
94.4 |
Test article |
2.00µg |
- |
497 |
258 |
252 |
255.0 |
51.3 |
98.1 |
Test article |
4.00µg |
- |
497 |
309 |
272 |
290.5 |
58.5 |
111.7 |
Test article |
6.00µg |
- |
497 |
220 |
226 |
223.0 |
44.9 |
85.8 |
Test article |
8.00 µg |
- |
497 |
13 |
20 |
16.5 |
3.3 |
6.3 |
Negative control |
0.00µg |
+ |
497 |
307 |
305 |
306.0 |
61.6 |
100.0 |
Solvent control DMSO |
0.00µg |
+ |
497 |
246 |
248 |
247.0 |
49.7 |
100.0 |
Positive control DMBA |
3.85µg |
+ |
497 |
270 |
249 |
259.5 |
52.2 |
105.1 |
Test article |
3.00µg |
+ |
497 |
256 |
271 |
263.5 |
53.0 |
86.1 |
Test article |
10.00µg |
+ |
497 |
227 |
234 |
230.5 |
46.4 |
75.3 |
Test article |
20.00µg |
+ |
497 |
253 |
253 |
253.0 |
50.9 |
82.7 |
Test article |
30.00µg |
+ |
497 |
244 |
259 |
251.5 |
50.6 |
82.2 |
Test article |
40.00µg |
+ |
497 |
258 |
299 |
278.5 |
56.0 |
91.0 |
Test article |
50.00µg |
+ |
497 |
217 |
240 |
228.5 |
46.0 |
74.7 |
*only colonies with more than 50 cells 7 days after seeding were scored
** PE absolute (value column 6/value column 3 x 100)
***PE relative (value column 6/value column 6 of corresponding control x 100)
Table II: Mutagenicity data, experiment I (part 1: cell survival)
Column |
Conc. Per ml |
S9 mix |
Number of cells per flask* |
Factor** calculated |
Cells seeded |
Cells*** survived |
|||||
seeded |
found |
mean |
|
|
|
||||||
I/II |
I |
II |
|
||||||||
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
|||
Negative control |
0.00µg |
- |
556 |
402 |
367 |
384.5 |
0.69 |
387000 |
267030 |
||
Negative control DMSO |
0.00µg |
- |
474 |
391 |
432 |
411.5 |
0.87 |
453000 |
394110 |
||
Positive control EMS |
0.60µg |
- |
567 |
457 |
431 |
444.0 |
0.78 |
375000 |
292500 |
||
Test article |
0.30µg |
- |
486 |
380 |
410 |
395.0 |
0.81 |
450000 |
364500 |
||
Test article |
1.00µg |
- |
526 |
300 |
335 |
317.5 |
0.60 |
480000 |
288000 |
||
Test article |
2.00µg |
- |
Culture was not continued |
|
|
|
|||||
Test article |
4.00µg |
- |
539 |
321 |
333 |
327.0 |
0.61 |
426000 |
259860 |
||
Test article |
6.00µg |
- |
Culture was not continued |
|
|
|
|||||
Test article |
8.00 µg |
- |
530 |
335 |
363 |
349.0 |
0.66 |
372000 |
245520 |
||
Negative control |
0.00µg |
+ |
568 |
380 |
373 |
376.5 |
0.66 |
450000 |
297000 |
||
Negative control DMSO |
0.00µg |
+ |
537 |
357 |
344 |
350.5 |
0.65 |
432000 |
280800 |
||
Positive control DMBA |
3.85µg |
+ |
479 |
307 |
323 |
315.0 |
0.66 |
423000 |
279180 |
||
Test article |
3.00µg |
+ |
579 |
374 |
331 |
352.5 |
0.61 |
402000 |
245220 |
||
Test article |
10.00µg |
+ |
461 |
324 |
330 |
327.0 |
0.71 |
450000 |
319500 |
||
Test article |
20.00µg |
+ |
Culture was not continued |
|
|
||||||
Test article |
30.00µg |
+ |
497 |
316 |
315 |
315.5 |
0.63 |
435000 |
274050 |
||
Test article |
40.00µg |
+ |
Culture was not continued |
|
|
|
|||||
Test article |
50.00µg |
+ |
486 |
264 |
297 |
280.5 |
0.58 |
435000 |
252300 |
||
*only colonies with more than 50 cells 7 days after seeding in normal medium were scored
** factor calculated (value column 6/value column 3)
***cells survived after plating in TG containing medium (value column 8 x value column 7)
Table III: Mutagenicity data, experiment I (part 2: mutation rates)
Column |
Conc per ml |
S9 mix |
Number of mutant colonies per flask* found after plating in TG medium |
Standard deviation |
Mutant** colonies per 106cells |
|||||
I |
II |
III |
IV |
V |
mean |
|||||
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
|
Negative control |
0.00µg |
- |
7 |
1 |
11 |
6 |
4 |
5.8 |
3.7 |
21.7 |
Negative control DMSO |
0.00µg |
- |
5 |
1 |
3 |
4 |
3 |
3.2 |
1.5 |
8.1 |
Positive control EMS |
0.60µg |
- |
103 |
83 |
93 |
91 |
80 |
90.0 |
9.1 |
307.7 |
Test article |
0.30µg |
- |
14 |
16 |
18 |
11 |
8 |
13.4 |
4.0 |
36.8 |
Test article |
1.00µg |
- |
3 |
6 |
7 |
4 |
10 |
6.0 |
2.7 |
20.8 |
Test article |
2.00µg |
- |
Culture was not continued |
|
|
|
|
|||
Test article |
4.00µg |
- |
4 |
8 |
8 |
6 |
9 |
7.0 |
2.0 |
26.9 |
Test article |
6.00µg |
- |
Culture was not continued |
|
|
|
|
|||
Test article |
8.00 µg |
- |
10 |
3 |
6 |
4 |
2 |
5.0 |
3.2 |
20.4 |
Negative control |
0.00µg |
+ |
5 |
8 |
5 |
8 |
11 |
7.4 |
2.5 |
24.9 |
Negative control DMSO |
0.00µg |
+ |
14 |
11 |
18 |
11 |
15 |
13.8 |
2.9 |
49.1 |
Positive control DMBA |
3.85µg |
+ |
131 |
123 |
126 |
139 |
101 |
124.0 |
14.2 |
444.2 |
Test article |
3.00µg |
+ |
4 |
10 |
5 |
12 |
10 |
8.2 |
3.5 |
33.4 |
Test article |
10.00µg |
+ |
5 |
6 |
8 |
3 |
5 |
5.4 |
1.8 |
16.9 |
Test article |
20.00µg |
+ |
Culture was not continued |
|
|
|
|
|||
Test article |
30.00µg |
+ |
6 |
5 |
6 |
8 |
8 |
6.6 |
1.3 |
24.1 |
Test article |
40.00µg |
+ |
Culture was not continued |
|
|
|
|
|||
Test article |
50.00µg |
+ |
5 |
9 |
8 |
11 |
5 |
7.6 |
2.6 |
30.1 |
*only colonies with more than 50 cells 8 days after seeding in TG medium were scored
**value column 8 x106/value column 9 (table I)
Table IV: toxicity data, experiment II
Column |
Conc. Per ml |
S9 mix |
Number of cells per flask* |
PE%** absolute |
PE%*** relative |
|||
seeded |
found |
mean |
||||||
I/II |
I |
II |
|
|||||
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
|
Negative control |
0.00µg |
- |
497 |
295 |
296 |
295.5 |
59.5 |
100.0 |
Solvent control DMSO |
0.00µg |
- |
497 |
289 |
262 |
275.5 |
55.4 |
100.0 |
Positive control EMS |
0.60µg |
- |
497 |
291 |
240 |
265.5 |
53.4 |
89.8 |
Test article |
0.30µg |
- |
497 |
353 |
277 |
315.0 |
63.4 |
114.3 |
Test article |
1.00µg |
- |
497 |
346 |
342 |
344.0 |
69.2 |
124.9 |
Test article |
2.00µg |
- |
497 |
280 |
287 |
283.5 |
57.0 |
102.9 |
Test article |
4.00µg |
- |
497 |
276 |
322 |
299.0 |
60.2 |
108.5 |
Test article |
6.00µg |
- |
497 |
371 |
341 |
356.0 |
71.6 |
129.2 |
Test article |
8.00 µg |
- |
497 |
310 |
315 |
312.5 |
62.9 |
113.4 |
Negative control |
0.00µg |
+ |
497 |
325 |
314 |
319.5 |
64.3 |
100.0 |
Solvent control DMSO |
0.00µg |
+ |
497 |
317 |
297 |
307.0 |
61.8 |
100.0 |
Positive control DMBA |
3.85µg |
+ |
497 |
191 |
206 |
198.5 |
39.9 |
64.7 |
Test article |
3.00µg |
+ |
497 |
303 |
284 |
293.5 |
59.1 |
91.9 |
Test article |
10.00µg |
+ |
497 |
315 |
267 |
291.0 |
58.6 |
91.1 |
Test article |
20.00µg |
+ |
497 |
299 |
297 |
298.0 |
60.0 |
93.3 |
Test article |
30.00µg |
+ |
497 |
289 |
303 |
296.0 |
59.6 |
92.6 |
Test article |
40.00µg |
+ |
497 |
282 |
272 |
277.0 |
55.7 |
86.7 |
Test article |
50.00µg |
+ |
497 |
263 |
294 |
278.5 |
56.0 |
87.2 |
*only colonies with more than 50 cells 7 days after seeding were scored
** PE absolute (value column 6/value column 3 x 100)
***PE relative (value column 6/value column 6 of corresponding control x 100)
Table V: mutagenicity data experiment II (part 1: cell survival)
Column |
Conc. Per ml |
S9 mix |
Number of cells per flask* |
Factor** calculated |
Cells seeded |
Cells*** survived |
|||||
seeded |
found |
mean |
|
|
|
||||||
I/II |
I |
II |
|
||||||||
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
|||
Negative control |
0.00µg |
- |
487 |
512 |
452 |
482.0 |
0.99 |
392000 |
388080 |
||
Negative control DMSO |
0.00µg |
- |
450 |
367 |
420 |
393.5 |
0.87 |
435000 |
378450 |
||
Positive control EMS |
0.60µg |
- |
515 |
434 |
448 |
441.0 |
0.86 |
444000 |
381840 |
||
Test article |
0.30µg |
- |
497 |
398 |
400 |
399.0 |
0.80 |
426000 |
340800 |
||
Test article |
1.00µg |
- |
476 |
375 |
401 |
388.0 |
0.82 |
498000 |
408360 |
||
Test article |
2.00µg |
- |
Culture was not continued |
|
|
|
|||||
Test article |
4.00µg |
- |
452 |
386 |
421 |
403.5 |
0.89 |
492000 |
437880 |
||
Test article |
6.00µg |
- |
Culture was not continued |
|
|
|
|||||
Test article |
8.00 µg |
- |
480 |
446 |
460 |
453.0 |
0.94 |
435000 |
408900 |
||
Negative control |
0.00µg |
+ |
505 |
377 |
397 |
387.0 |
0.77 |
369000 |
284130 |
||
Negative control DMSO |
0.00µg |
+ |
492 |
338 |
436 |
387.0 |
0.79 |
306000 |
241740 |
||
Positive control DMBA |
3.85µg |
+ |
494 |
333 |
334 |
333.5 |
0.68 |
396000 |
269280 |
||
Test article |
3.00µg |
+ |
495 |
369 |
393 |
381.0 |
0.77 |
402000 |
309540 |
||
Test article |
10.00µg |
+ |
510 |
346 |
308 |
327.0 |
0.64 |
378000 |
241920 |
||
Test article |
20.00µg |
+ |
Culture was not continued |
|
|
||||||
Test article |
30.00µg |
+ |
533 |
245 |
283 |
264.0 |
0.50 |
387000 |
193500 |
||
Test article |
40.00µg |
+ |
Culture was not continued |
|
|
|
|||||
Test article |
50.00µg |
+ |
493 |
407 |
367 |
387.0 |
0.78 |
384000 |
299520 |
||
*only colonies with more than 50 cells 7 days after seeding in normal medium were scored
** factor calculated (value column 6/value column 3)
***cells survived after plating in TG containing medium (value column 8 x value column 7)
See Table VI in overall remarks.
Taking into account the mutation rates found in the groups treated with the test article compared to the negative and solvent controls it can be concluded that no biologically relevant increase of point mutations was observed. The test article did not induce reproducible concentration-related increase in the mutant colony numbers. The mutant values of the groups treated with the test article were in the range of the negative controls.
In this study in both experiment (with and without S9 mix) the range of the negative controls was from 0.7 up to 49.1 mutants per 1000000 cells, the range of the groups treated with the test article was from 0.5 up to 36.8 mutants per 1000000 cells.
EMS (0.6 mg(ml) and DMBA (3.85 µg/ml) were used as positive controls and showed a distinct increase in induced mutant colonies.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
The substance did not induce micronuclei in bone marrow cells when tested to the maximum tolerated doses of 1000 mg/kg/day in male and 800 mg/kg/day in female CD rats using a 0 h + 24 h oral dosing and 48 h sampling regimen.
The substance was negative for inducing genotoxicity in the liver, duodenum, and glandular stomach in the In Vivo Mammalian Alkaline Comet Assay employing 5 Sprague Dawley rats/sex/dose in corn oil by gavage at dose levels of 0, 200, 400 or 800 mg/kg bw/day (with a mean peak plasma concentration of 1901 μg/mL) once daily for 2 consecutive days (20 hours apart).
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- micronucleus assay
- Specific details on test material used for the study:
- - Name of test material (as cited in study report): TK 12891/1 (also known as 2,4-di-tert.-butylphenol)
- Physical state: yellow solid
- Analytical purity: 99.1%
- Supplier: Sigma-Aldrich
- Lot/batch No.: S43419-228
- Expiration date of the lot/batch: 24 July 2009
- Storage condition of test material: stored at ambient temperature in the dark when not in use - Species:
- rat
- Strain:
- other: CD rats
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River UK
- Weight at study initiation: mean(males): 266 - 312 g; mean(females): 160 - 193 g
- Housing: in sets of twos and threes
- Diet: SDS Rat and Mouse Maintenance Diet No. 1 which was obtained from Special Diet Services Limited, England. (ad libitum)
- Water: tap water (ad libitum)
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 22
- Humidity (%): 46 - 87
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12
- Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: corn oil
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The dose volume used for both the control and test item treated animals was a constant 10 mL/kg body weight.
- Duration of treatment / exposure:
- Bone marrow samples were taken 48 h after the initial 0 h dose.
- Frequency of treatment:
- Twice (Rats were dosed at 0 h and 24 h.)
- Post exposure period:
- Bone marrow samples were taken 48 h after the initial 0 h dose.
- Dose / conc.:
- 200 mg/kg bw/day (nominal)
- Remarks:
- Females
- Dose / conc.:
- 400 mg/kg bw/day (nominal)
- Remarks:
- Females
- Dose / conc.:
- 800 mg/kg bw/day (nominal)
- Remarks:
- Females
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Remarks:
- Males
- No. of animals per sex per dose:
- - vehicle control: 5 males and 5 females;
- low/mid dose: 5 females each;
- high dose: 10 males and 10 females (The high dose group of rats consisted of an increased group size of 10 males and 10 females of which 5 males and 5 females provided the regular assessment base. The additional rats were processed in normal fashion and the slides labelled with the original animal number. The slides from this spare group were kept as a contingency in case of unscheduled deaths or potential sex differences. In the event of death, the first available animal in the relevant contingency group replaced the missing animal. Preparations were made from remaining contingency group animals and the slides were kept as spares.);
- positive control: 5 males; - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- cyclophosphamide
- Doses / concentrations: 50 mg/kg bw/day (Cyclophosphamide was prepared fresh as a 5 mg/mL solution in distilled water. It was administered to the positive control animals in dose volumes of 10 mL/kg to give the required target dose of 50 mg/kg) - Tissues and cell types examined:
- bone marrow cells
- Details of tissue and slide preparation:
- DETAILS OF SLIDE PREPARATION:
A drop of the suspension including bone marrow cells was placed at one end of the slide and a smear made by drawing the top of a Pasteur pipette horizontally along the slide. Two slides were prepared from each tube/animal. The smear was left to air dry, fixed in methanol for ca 5 min and then immersed for 15 min in 15% Giemsa stain, prepared in tap water, to give optimum erythrocyte discrimination. The stained smears were finally rinsed in distilled water for ca 1 min and left to air dry overnight. Permanent slide preparations were made by sealing coverslips onto the glass slides using DPX mounting medium.
METHOD OF ANALYSIS:
The better of the 2 prepared slides was selected for examination and the coded slides assessed blind by the same operator. At least two thousand (2000) polychromatic erythrocytes (PCE) per animal were scored for micronuclei and the frequency of micronucleated cells (MN-PCE) determined. As a control against inclusion of artefacts, or action of a mutagen on the G2 and/or mitotic phase of the cell cycle, the numbers of micronucleated normochromatic erythrocytes (MN-NCE) in mature red blood corpuscles were also recorded (Maier and Schmid, 1976; Hamoud et al, 1989). In addition, scored micronuclei were assigned on the basis of size into small or large categories, historically defined as micronuclei occupying less or more than 25% of the visible cellular area. This classification provided a non-specific measure of compound induced spindle dysfunction, as large micronuclei appear to derive from lagging chromosomes caused by damage to the mitotic apparatus during bone marrow erythropoiesis (Yamamoto and Kikuchi, 1980; Vanderkerken et al, 1989). The PCE/NCE ratio, a measure of any induced systemic toxicity, was determined by counting a minimum total of 1000 erythrocytes (PCE + NCE) per marrow preparation.
- Positive Response:
The test would be judged positive if an increase in the number of micronucleated polychromatic erythrocytes (MN-PCE) was obtained for one or more of the test item treated dose groups. That is, an increase greater than 10% over the expected historical control ranges for a group of animals. The increase observed should be biologically relevant and statistically significant relative to concurrent and historical control frequencies for MN-PCE and/or MN-NCE induction.
- Inconclusive Response:
The test would be considered inconclusive if the levels of MN-PCE within any one dose group were increased above the established historical control frequencies for MN-PCE induction, but not high enough to meet the criteria for a positive response. That is an increase up to 10% over the maximum negative control frequency for a group of animals. - Evaluation criteria:
- Acceptance Criteria:
- The prepared slides had uniform staining properties and sufficient number of PCE cells present to allow accurate micronucleus determination.
- The assay was considered acceptable as the MN-PCE frequencies for the vehicle control dosed rats were within the expected historical range. The ranges are defined in accordance with Charles River Laboratories experience of the bone marrow micronucleus test using CD rats.
- An adequate positive control response for at least 2 animals and the dose group as a whole.
Evaluation Criteria:
The average micronucleus incidence in vehicle control dosed and untreated CD rats, has in this laboratory been determined as 0.06±0.05% , a range of 0.01-0.13% per group of 5-6 animals and 0.03-0.11% per group of 10-12 animals. This frequency is in agreement with published data for micronucleus tests with CD rats (Tamura et al, 1990; Salamone and Mavournin, 1994). These historical data have been used in the evaluation of response in this test.
- Negative Response:
The test would be judged negative if no biologically relevant increases in the numbers of MN-PCE were observed, relative to the concurrent and established historical control frequencies for MN-PCE induction. No statistical analysis will be performed if the levels of MN-PCE induction fell within the determined historical control frequencies. A similar biological approach to the data, which avoids the need for statistical evaluations, has recently been described (Ashby and Tinwell, 1995). Variations in the MN-NCE frequencies and PCE/NCE ratios will also not be analysed statistically, unless clearly different from concurrent control values. - Statistics:
- Not required
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- There were indications of bone marrow toxicity in the high dose males (PCE/NCE ratio of 0.43 compared to 0.56 in vehicle control group).
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
As toxicity information on the test item was available from the Sponsor, a limit toxicity test (3 males/3 females) was conducted prior to the micronucleus test to establish a suitable dose range for the micronucleus experiment. The limit toxicity study used a starting dose of 1600 mg/kg. Mortality occured at the ranger-finder doses of 1000, 1200, 1400 and 1600 mg/kg bw. At 1600 mg/kg bw each one of three males and females died on day 3 after dosing. Based on the findings of the toxicity study, the maximum tolerated doses were judged to be in the region of 1000 mg/kg/day for males and 800 mg/kg/day for females.
RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay):
- Induction of micronuclei (for Micronucleus assay): in the range of the negative control
- Ratio of PCE/NCE (for Micronucleus assay): There were indications of bone marrow toxicity in the high dose males (PCE/NCE ratio of 0.43 compared to 0.56 in vehicle control group). - Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 21 October 2020 - 10 February 2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
- Deviations:
- yes
- Remarks:
- All of deviations were considered minor and of no impact to the integrity of the study.
- GLP compliance:
- yes
- Type of assay:
- mammalian comet assay
- Specific details on test material used for the study:
- Name of test material (as cited in study report): 2,4-di-tert-butylphenol (2,4-DTBP)
Batch No.: 200144687 - Species:
- rat
- Strain:
- Sprague-Dawley
- Details on species / strain selection:
- As required by the test guideline. Sprague Dawley rats were selected for this study to be consistent with most in vivo comet studies conducted at the test facility.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories (CRL), Raleigh, NC
- Age at study initiation: 8 weeks
- Weight at study initiation: 176.06 - 339.35 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: high polysulfone cages with absorbent bedding - single housed
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 1 day
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 – 24°C
- Humidity (%): 21 - 43%
- Air changes (per hr): 70
- Photoperiod (hrs dark / hrs light): 12 hours of light and 12 hours of dark - Route of administration:
- oral: gavage
- Vehicle:
- Corn oil
- Details on exposure:
- The substance and vehicle control dose formulations were prepared once for each experiment within 8 days of dosing and stored refrigerated and protected from light. Dose calculations were not adjusted to account for the test substance purity. The dose volume for each dose administration was 10 mL/kg.
- Duration of treatment / exposure:
- 2 days
- Frequency of treatment:
- Once daily - 20±0.5 hours apart
- Dose / conc.:
- 800 mg/kg bw (total dose)
- Dose / conc.:
- 400 mg/kg bw (total dose)
- Dose / conc.:
- 200 mg/kg bw (total dose)
- Dose / conc.:
- 0 mg/kg bw (total dose)
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Ethyl methanesulfonate (EMS) prepared fresh in deionized water (dH2O) on the day of dosing and administered at a concentration of 300 mg/kg BW. The positive control group received a single oral administration of EMS 4 hours before necropsy and at a dose volume of 10 mL/kg.
- Tissues and cell types examined:
- Liver, central portion of the left lateral lobe, Duodenum, portion most proximal to stomach, Stomach, portion of glandular region, Ovary, half of one gonad (females only), Testis, central portion of one gonad (males only)
- Details of tissue and slide preparation:
- Based on the results from the dose range finder experiment, 800 mg/kg bw was selected as the maximum tolerable dose for both male and female rats in the comet assay. For each sample, four comet slides (replicates B-E) were prepared and after at least one hour in lysis, at least two replicate slides per tissue per sex (liver, duodenum, glandular stomach, ovary, testis) were electrophoresed under alkaline (pH >13) conditions. Prior to electrophoresis, slides were rinsed with 0.4M Tris (pH 7.5) and submerged in alkaline electrophoresis buffer (300 mM NaOH, 1 mM Na2EDTA; pH>13) for 20 minutes at 4.3 to 5.0°C to unwind the DNA. After unwinding, slides were electrophoresed at 4.4 to 4.8°C for 40 minutes at a constant voltage of 0.7V/cm. At the start of electrophoresis, the buffer level was adjusted as necessary to achieve a starting current of 300±10 mA. After electrophoresis, the electrophoresed slides were neutralized with 0.4M Tris buffer (pH 7.5) for 5 minutes, dipped in ethanol for 5 minutes, air dried and stored at room temperature. Slides were stained with SYBR Gold™ stain and unless precluded by poor cell density and/or poor sample/slide quality, 150 cells per sample (75 cells per slide, if possible) were scored using the Komet© Image Analysis System with Luca R camera (Andor Technology, Northern Ireland).
- Evaluation criteria:
- In studies where no statistically significant (p<0.05) response is detected at any substance dose concentration, the concurrent positive control must induce a statistically significant increase in DNA damage as determined by the %Tail when compared to the concurrent vehicle control.
The concurrent negative control must be considered acceptable for addition to the Helix3 historical control database by providing a sufficient dynamic range to detect a positive effect.
The test substance may be classified as positive for inducing genotoxicity if the following criteria are met:
a. a statistically significant increase (indicative of strand breaks) or decrease (indicative of crosslinks) in DNA migration is detected at one or more dose concentrations and
b. a statistically significant dose dependent response is detected in the same sample type
A test substance may be classified as equivocal for inducing genotoxicity if either criteria (a) or (b) are met, but not both. If an increase in cytotoxicity is detected in the same tissue and dose concentration(s) at which DNA damage is significantly affected, cytotoxicity may be considered a confounding factor in the determination of genotoxicity and a repeat test with the same and/or lower doses may be recommended to verify the presence of genotoxicity in the absence of cytotoxicity. If neither criteria (a) nor (b) are met and direct or indirect evidence of target organ exposure can be demonstrated, the test substance may be classified as negative for inducing genotoxicity. Since OECD 489 acknowledges that the etiology of ghost cells is uncertain, no attempt was made to interpret it or otherwise use it for the interpretation or qualification of the comet assay results. - Statistics:
- Using individual animal data from each comet assay experiment, statistical analysis with Analyse-It (Analyse-It Software, UK) was conducted on the extent of DNA migration as determined by the %Tail and on the %LMW. To be consistent with historical control data, the individual animal mean %Tail values were calculated as the mean of the total cells. A 95% confidence Interval (CI) was used for all statistical analyses. The Shapiro-Wilk test was conducted on the concurrent vehicle control dose group of the same sex to determine the normality of the data distribution. Based on the normality of the data, the appropriate pairwise comparisons were used to compare each dose to the concurrent vehicle control and the appropriate trend test was used to determine the presence of a dose-dependent response.
Normally distributed data was analyzed using two-tailed Fisher F-tests followed by a one-tailed Student’s t-test for dose groups with equal variances or a one-tailed Welch t-test for dose groups with unequal variances. The two-tailed Line Fit trend test was conducted.
Data that was not normally distributed was analyzed using one-tailed Wilcoxon-Man-Whitney tests. A two-tailed Kendall rank trend test was conducted. - Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- Please see details below.
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
The mean analyzed concentrations of 2,4-DTBP in all test substance dose group formulations used during the comet assay fulfilled the acceptance criterion (average percent recovery of 100±15% with ≤10% RSD), with an average percent recovery within 3.5% of their nominal values. No measurable concentrations of 2,4-DTBP were found in the vehicle control formulations.
- Dose range: 250, 500 and 1000 mg/kg bw
- Clinical signs of toxicity in test animals: male animals in the 250, 500 and 1000 mg/kg dose groups experienced lethargy, loose stool/diarrhea, and porphyrin staining. One male animal in the 500 mg/kg dose group was humanely sacrificed prior to the second dose administration due to excessive weight loss (>10%). Compared to the vehicle control dose group, female animals in the 800 mg/kg dose groups experienced lethargy, loose stool/diarrhea, and porphyrin staining. One female animal in the 100 mg/kg dose group was humanely sacrificed immediately following the first dose administration due to aspiration of the stomach contents caused by gastroesophageal reflux and was replaced with an extra animal. During necropsy, the ovary of one female animal in the 400 mg/kg dose group was noted as very pale in color. Compared to the vehicle control dose group, there was a dose-related decrease in average body gain in both sexes.
RESULTS OF DEFINITIVE STUDY
2,4-DTBP was detected in the plasma samples from all animals dosed with 2,4-DTBP. No measurable concentrations of 2,4-DTBP were detected in any of the vehicle control samples. At the time comet samples were collected, the mean plasma concentration of the highest dose tested (800 mg/kg) was 1075 ng/mL for males and 2727 ng/mL for females. Details are presented below.
- Clinical signs of toxicity in test animals: Compared to the vehicle control dose group, male animals in the 800 mg/kg bw dose groups experienced loose stool/diarrhea. During necropsy, one animal in the vehicle control group was noted to have only one testis present which was ~75% smaller than normal with dense tissue and no internal contents present. One animal in the 800 mg/kg dose group was noted to have testes ~25-50% smaller than normal. This issue was discussed with the Facility Vet and it was determined that it is not atypical for 8–9-week-old rats to have testes that are underdeveloped. Compared to the vehicle control dose group, there was a dose-related decrease in average bodyweight gain (please refer to table below). Compared to the vehicle control dose group, female animals in the 800 mg/kg bw dose groups experienced loose stool/diarrhea, dehydration, and decreased movement. Compared to the vehicle control dose group, there was a dose-related decrease in average bodyweight gain (please refer to table below). - Conclusions:
- Under the experimental conditions of the study, 2,4-DTBP was negative for inducing genotoxicity in the liver, duodenum, and glandular stomach of treated male and female animals up to the maximum tolerated dose of 800 mg/kg with a mean peak plasma concentration of 1901 µg/mL.
- Executive summary:
In a genotoxicity study performed in compliance with OECD Test No. 489: In Vivo Mammalian Alkaline Comet Assay 2,4-DTBP was administered to 5 Sprague Dawley rats/sex/dose in corn oil by gavage at dose levels of 0, 200, 400 or 800 mg/kg bw/day once daily for 2 consecutive days (20 hours apart).
Under the experimental conditions of the study, 2,4-DTBP was negative for inducing genotoxicity in the liver, duodenum, and glandular stomach of treated male and female animals up to the maximum tolerated dose of 800 mg/kg bw/day with a mean peak plasma concentration of 1901 μg/mL.Based on the decreased body weight gains in the 400 and 800 mg/kg bw dose groups, the statistically significant dose-related decrease in duodenum and glandular stomach DNA migration in female rats is most likely indicative of cell loss due to a cytotoxic response. The possibility of crosslink inductions reducing DNA migration in these dose groups is unlikely due to the absence of a concomitant decrease in %LMW at the same doses a decrease in DNA migration was detected
Clinical signs of toxicity included loose stool/diarrhea in male animals in the 800 mg/kg bw dose groups. There was a dose-related decrease in average bodyweight gain at the top two doses in males. Female animals in the 800 mg/kg bw dose groups experienced loose stool/diarrhea, dehydration, and decreased movement. Compared to the vehicle control dose group, there was a dose-related decrease in average bodyweight gain at the top dose.
This genotoxicity study in the rat is acceptable and satisfies the guideline requirement for a Comet assay (OECD Test No. 489: In Vivo Mammalian Alkaline Comet Assay) in rats. To conclude, under the experimental conditions of the study, 2,4-DTBP was negative for inducing genotoxicity in the liver, duodenum, and glandular stomach of treated male and female animals up to the maximum tolerated dose of 800 mg/kg with a mean peak plasma concentration of 1901 µg/mL.
Referenceopen allclose all
| Test group | Sex | No. of rats scored | erythrocytes | ||||
| Normochromatic cells (NCE) | Polychromatic cells (PCE) | PCE/NCE Mean +/- S.D. | |||||
| No. of MN-NCE | PCE analysed | No. of MN-PCE | % MN-PCE | ||||
| vehicle (20 ml corn oil kg/day) | male | 5 | 8 | 10004 | 7 | 0.07 | 0.53 +/- 0.06 |
| female | 5 | 4 | 10008 | 4 | 0.04 | 0.58 +/- 0.05 | |
| male/female | 10 | 12 | 20012 | 11 | 0.05 | 0.56 +/- 0.06 | |
| 200 mg /kg/day | female | 5 | 1 | 10009 | 6 | 0.06 | 0.67 +/- 0.12 |
| 400 mg /kg/day | female | 5 | 3 | 10012 | 5 | 0.05 | 0.58 +/- 0.13 |
| 1000 mg /kg/day | male | 5 | 9 | 10011 | 3 | 0.03 | 0.43 +/-0.08 |
| 800 mg /kg/day | female | 5 | 2 | 10008 | 5 | 0.05 | 0.59 +/- 0.12 |
| 50 mg Cyclophosphamide /kg/day | male | 5 | 53¿ | 10002 | 189¿ | 1.89 | 0.34 +/- 0.07 |
| PCE = polychromatic erythrocyts | |||||||
| MN-PCE = micronucleated PCE | |||||||
| NCE = normochromatic erythrocyts | |||||||
| MN-NCE = micronucleated NCE | |||||||
| ¿= positive response in PCE | |||||||
| ¿ = evident response in NCE | |||||||
No animal deaths occurred following dosing. Clinical signs of hunched, subdued behaviour, wet around anus, wet staining pergenital, wet faeces, piloerection, salivation red discharge (nose), laboured breathing and staggering were observed. There was no indication that the test item induced bone marrow micronuclei in the treated rats. The highest MN-PCE frequency recorded for the test item was in the low dose females where an incidence of 0.06% was observed. There were indications of bone marrow toxicity in the high dose males (PCE/NCE ratio of 0.43 compared to 0.56 in vehicle control group).
Bioanalytical Concentration Analysis
| Sex | Dose (mg/kg bw) | Mean Plasma Concentrations of 2,4-DTBP (ng/mL) |
| Male | 200 | 493 |
| Male | 400 | 545 |
| Male | 800 | 1075 |
| Female | 200 | 748 |
| Female | 400 | 755 |
| Female | 800 | 2727 |
DRF Bodyweight Summary Data
| Dose (mg/kg/day) | Average Weight Gain - Male (g) | Average Weight Gain - Female (g) |
| 0 (vehicle) | 11.08 | 3.68 |
| 125 | 8.61 | 5.43 |
| 250 | 13.40 | 3.69 |
| 500 | -5.01 | 6.88 |
| 1000 | -15.78 | -3.64 |
Comet Assay Bodyweight Summary Data
| Dose (mg/kg bw/day | Average Weight Gain - Males (g) | Average Weight Gain - Females (g) |
| 0 (vehicle) | 8.72 | 6.95 |
| 200 | 10.64 | 4.57 |
| 400 | 1.13 | 4.29 |
| 800 | 1.13 | -5.23 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Justification for classification or non-classification
Based on the findings of a reliable in vitro and in vivo genotoxicity studies conducted on the substance and in a read-across substance, classification of the substance is not justified.
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