Registration Dossier
Registration Dossier
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EC number: 271-668-0 | CAS number: 68603-62-3
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Reverse gene mutation assay: Negative (non-mutagenic); OECD 471; Dreher, 2017
Mammalian cell cytogenicity assay: Negative (non-clastogenic); OECD 473; Lloyd, 2017
Mammalian cell gene mutation assay: Negative (non-mutagenic); OECD 476; Lloyd, 2018
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:
- 13 February - 06 November 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15-25 ºC, protected from light
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: Soluble at 573.6 mg/mL in DMSO
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test article stock solutions were prepared by formulating the test article under subdued lighting in DMSO with the aid of vortex mixing and warming at 37 ̊C, to give the maximum required treatment concentration. Subsequent dilutions were made using DMSO.
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: 50 mg/mL prepared as the solution for the highest tested concentration. Subsequent dilutions were made using DMSO.
- Final preparation of a solid: N/A
FORM AS APPLIED IN THE TEST (if different from that of starting material) : Appled as a liquid
TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) : N/A
OTHER SPECIFICS: N/A - 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
- Test concentrations with justification for top dose:
- A maximum concentration of 5000 μg/plate was selected for Experiment 1, to ensure that the initial treatments were performed up to this maximum recommended concentration according to current regulatory guidelines.
Experiment 1 (with and without S9 mix): 5000, 1600, 500, 160, 50, 16, 5 µg/plate
Experiment 2 (without S9 mix): 1600, 800, 400, 200, 100, 50 µg/plate
Experiment 2 (with S9 mix): 1600, 800, 400, 200, 100, 50 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Maron, D., Katzenellenbogen, J., and Ames, B.N. (1981). Compatibility of Organic Solvents
with the Salmonella/Microsome Test. Mutation Res., 88, 343-350. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO; (Experiment 1: batch number STBG36578V, supplied by Sigma-Aldrich Chemical Company, no expiry date specified by the supplier; Experiment 2: batch number STBG4158V, supplied by Sigma-Aldrich Chemical Company, no expiry date specified by the supplier
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- benzo(a)pyrene
- mitomycin C
- other: 2-aminoanthracene (AAN)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): 10E8 - 10E9 cells/mL
DURATION
- Preincubation period: 20 mins (Experiment 2, with S9 mix only)
- Exposure duration: 2-3 days
- Expression time (cells in growth medium): 2-3 days
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A
SELECTION AGENT (mutation assays): N/A
SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for cytogenetic assays): N/A
NUMBER OF REPLICATIONS: 3
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: N/A
NUMBER OF CELLS EVALUATED: N/A
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): N/A
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: N/A
DETERMINATION OF CYTOTOXICITY
- Method: thinning/ absence of bacteria lawn
OTHER EXAMINATIONS:
- Determination of polyploidy: N/A
- Determination of endoreplication: N/A
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): N/A
- OTHER: N/A - Rationale for test conditions:
- The study was based on the in vitro technique described by Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item. These strains have a deleted excision repair mechanism which makes them more sensitive to various mutagens and they will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium. Using these strains of Salmonella typhimurium revertants may be produced after exposure to a chemical mutagen, which have arisen as a result of a base-pair substitution in the genetic material (miscoding) or as a frameshift mutation in which genetic material is either added or deleted
- Evaluation criteria:
- For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values.
2. Any observed response was reproducible under the same treatment conditions.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if neither of the above criteria were met. - Statistics:
- The statistical significance of results was analysed using Dunnett’s test to aid in the evaluation of potential positive responses.
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- 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
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Conclusions:
- The test item was considered to be non-mutagenic under the conditions of the test.
- Executive summary:
OECD 471 (2017) - In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537 and TA102 of S. typhimuriumwere exposed to Amines, C12-14-branched alkyl dodecylbenzenesulfonates (1:1) in DMSO at concentrations of 5, 16, 50, 160, 500, 1600 and 5000 µg/plate a screening experiment followed by concentrations of 50, 100, 200, 400, 800 and 1600 µg/plate in a secondary, pre-incubation, experiment. Both tests were conducted in the presence and absence of S9 mix.
The test item was tested up to cytotoxic and precipitating concentrations. The positive controls induced the appropriate responses in the corresponding strains.
There was no evidence of induced mutant colonies over background in the test item treated colonies.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 04 April - 01 December 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Study was conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Guideline 40 CFR 799.9537 TSCA: in vitro mammalian chromosome aberration test (EPA, 2011)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15-25 ºC, protected from light
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: Preliminary solubility data indicated that the test article was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at a concentration of at least 538.99 mg/mL. The solubility limit in culture medium was in the range of 168.4 to 336.9 μg/mL, as indicated by precipitation at the higher concentration which persisted for approximately 22 hours after test article addition, with warming at 37 °C.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test article stock solutions were prepared by formulating the test article under subdued lighting in DMSO, with the aid of vortex mixing (Range-Finder and Micronucleus Experiment) and warming at 37°C (Range-Finder only) to give the maximum required concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 3.5 hours of initial formulation.
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: 120 mg/mL, with further dilutions prepared in DMSO
- Final preparation of a solid: N/A
FORM AS APPLIED IN THE TEST (if different from that of starting material) : applied as a liquid
TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) : N/A
OTHER SPECIFICS: N/A - Species / strain / cell type:
- lymphocytes: human
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Blood from three healthy, non-smoking female volunteers from a panel of donors. No donor was suspected of any virus infection or exposed to high levels of radiation or hazardous chemicals. All donors are non-smokers and are not heavy drinkers of alcohol. Donors were not taking any form of medication (contraceptive pill excluded).
- Suitability of cells: Screened as described above.
- Cell cycle length, doubling time or proliferation index: The measured cell cycle time of the donors used at Covance, Harrogate falls within the range 13±2 hours.
- Sex, age and number of blood donors if applicable: Female; aged 23-33
- Whether whole blood or separated lymphocytes were used if applicable: Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL of pooled heparinised blood into 8.5 mL pre-warmed HEPES-buffered RPMI medium containing 10 % (v/v) heat inactivated foetal calf serum and 0.52 % penicillin / streptomycin (in an incubator set to 37±1 °C), so that the final volume following addition of S-9 mix or KCl and the test article in its chosen vehicle was 10 mL. The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2 % of culture to stimulate the lymphocytes to divide. Blood cultures were incubated at 37±1 °C for approximately 48 hours and rocked continuously.
- Number of passages if applicable: 1
- Methods for maintenance in cell culture if applicable: Described above
- Modal number of chromosomes: 46
- Normal (negative control) cell cycle time: 13±2 h
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: HEPES-buffered RPMI medium containing 10% (v/v) heat inactivated foetal calf serum and 0.52% penicillin / streptomycin. The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No
- Periodically checked for karyotype stability: No
- Periodically 'cleansed' against high spontaneous background: No - Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- The solubility limit of the test item in the culture medium was in the range of 168.4-336.9 µg/mL. 1200 µg/mL was selected as the top concentration in order to perform the test up to precipitating concentrations (OECD 473).
See Table 1 for concentration ranges tested. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Maron, D., Katzenellenbogen, J., and Ames, B.N. (1981). Compatibility of Organic Solvents
with the Salmonella/Microsome Test. Mutation Res., 88, 343-350. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- See materials and methods section below.
- Evaluation criteria:
- For valid data, the test article was considered to induce clastogenic events if:
1. Compared to the concurrent negative control a statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) at one or more concentrations is observed (p ≤ 0.05).
2. The incidence of cells with structural aberrations (excluding gaps) at such a concentration exceeds the normal range in both replicate cultures and falls outside the distribution of the historical negative control data.
3. A concentration-related increase in the proportion of cells with structural aberrations (excluding gaps) is observed (positive trend test).
The test article was considered positive in this assay if all of the above criteria were met. - Statistics:
- After completion of scoring and decoding of slides the numbers of aberrant cells in each culture were categorised as follows:
1. Cells with structural aberrations including gaps
2. Cells with structural aberrations excluding gaps
3. Polyploid or endoreduplicated cells.
The totals for category 2 in vehicle control cultures were compared with the 95th percentile of the current historical vehicle control (normal) ranges to determine whether the assay was acceptable or not (see Acceptance criteria). The proportion of cells with structural chromosome aberrations excluding gaps were compared with the proportion in vehicle controls by using Fisher’s exact test (Richardson et al., 1989). In addition, a Cochran-Armitage Trend Test was performed to aid determination of concentration response relationships. Probability values of p≤0.05 were accepted as significant. The proportions of cells in categories 1 and 3 were examined in relation to historical vehicle control range.
The proportions of aberrant cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test (Richardson et al., 1989). Probability values of p≤0.05 were accepted as significant. - Key result
- Species / strain:
- hepatocytes: Human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- See results section below.
- Conclusions:
- The test item did not induce structural chromosome aberrations in cultured human peripheral blood lymphocytes when tested up to the limit of cytotoxicity for 3+17 hours in the absence and presence of a rat liver metabolic activation system (S-9) and for 20+0 hours in the absence of S-9
- Executive summary:
OECD 473 (2017) - In a mammalian cell cytogenetics assay (in vitro chromosome aberration, OECD 473), primary lymphocyte cultures were exposed to Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1) at concentrations of 0, 10, 20, 40, 60, 80, 90, 100, 110, 120, 130, 140, 150 and 175 µg/mL for 3 h with and without metabolic activation. Additionally, a continuous exposure of 24 h was tested without metabolic activation at test item concentrations of 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 80, 100 µg/mL.
Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1) was tested up to precipitating and cytotoxic concentrations of 155.5 and 55.99 µg/mL for the 3 h and 20 h exposures, respectively. Positive controls induced the appropriate response.
There was no evidence (or a concentration related positive response) of chromosome aberration induction over background values at the highest tested concentration.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline In vitro Mammalian Chromosome Aberration Test (OECD 473) in human lymphocyte cells.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 11 September 2017 -
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Study was conducted in accordance with International guidleines and in accordance with GLP. All guideline validity criteria were met.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Specific details on test material used for the study:
- RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15-25 ºC, protected from light
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: 583.99 µg/mL in DMSO, assumed stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test article stock solutions were prepared by formulating the test article under subdued lighting in DMSO, with the aid of vortex mixing and warming at 37°C, to give the maximum required concentration. Subsequent dilutions were made using DMSO.
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: Test article stock solutions were prepared by formulating the test article under subdued lighting in DMSO, with the aid of vortex mixing and warming at 37°C, to give the maximum required concentration. Subsequent dilutions were made using DMSO.
- Final preparation of a solid: N/A
FORM AS APPLIED IN THE TEST (if different from that of starting material) : Applied as a liquid
TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) : N/A
OTHER SPECIFICS: N/A - Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Dr Donald Clive, Burroughs Wellcome Co. Cells
- Suitability of cells: Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free.
- Cell cycle length, doubling time or proliferation index: Not reported
- Sex, age and number of blood donors if applicable: N/A
- Whether whole blood or separated lymphocytes were used if applicable: N/A
- Number of passages if applicable: Not reported
- Methods for maintenance in cell culture if applicable: N/A
- Modal number of chromosomes: N/A
- Normal (negative control) cell cycle time: Not reported
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI medium with 5±1 % CO2
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Supplied mycoplasm-free
- Periodically checked for karyotype stability: Not reported
- Periodically 'cleansed' against high spontaneous background: Not reported - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Prelim: 18.75, 37.5, 75, 150, 300, 600 µg/mL (600 µg/mL chosen as a concentration beyond the limit of solubility in DMSO and in the culture medium i.e. precipitating concentration)
Main: 5, 10, 20, 30, 40, 50, 55, 60, 65, 70, 80, 100 µg/mL (100 µg/mL chosen based on the results of the range-finder where excessive toxicity was observed in the top 2 concentrations) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Guideline recommended solvent vehicle - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- benzo(a)pyrene
- Details on test system and experimental conditions:
- See materials and methods section below
- Evaluation criteria:
- For valid data, the test article was considered to be mutagenic in this assay if:
1. The MF at one or more concentrations was significantly greater than that of the negative control (p ≤ 0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p ≤ 0.05)
3. If both of the above criteria were fulfilled, the results should exceed the upper limit of the last 20 studies in the historical negative control database (mean MF +/ 2 standard deviations.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. - Statistics:
- Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No changes observed
- Effects of osmolality: No changes observed
- Evaporation from medium: Not reported
- Water solubility: Not reproted
- Precipitation: Tested up to precipitating levels
- Definition of acceptable cells for analysis: Not reported
- Other confounding effects: No
RANGE-FINDING/SCREENING STUDIES: In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 18.75 to 600 µg/mL (limited by precipitation of the formulated test article in culture medium). The highest concentration to give ≥10 % relative survival (RS) was 37.5 µg/mL, which gave 66 % and 62 % RS in the absence and presence of S-9, respectively.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges.
vehicle control: 4.67 (-S9) and 4.92 (+S9); historical range: 1.15 - 8.20 (-S9) and 2.42 - 7.42 (+S9)
positive control (NQO 0.15 µg/mL, -S9): 45.51; historical range: 4.04 - 86.97
positive control (NQO 0.20 µg/mL, -S9): 49.20; historical range: 7.90 - 90.49
positive control (B[a]P 2 µg/mL, +S9): 38.37; historical range: 4.06 - 72.69
positive control (B[a]P 3 µg/mL, +S9): 48.54; historical range: 14.95 - 82.13
(Values are mutants per 10xE6 viable cells) - Conclusions:
- It is concluded that Amines, C12-14-branched alkyl dodecylbenzenesulfonates (1:1) did not induce biologically relevant increases in mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.
- Executive summary:
OECD 476 (2018) - In a mammalian cell gene mutation assay (HPRT locus of L5178Y), mouse lymphoma cells cultured in vitro were exposed to Amines, C12-14-branched alkyl dodecylbenzenesulfonates (1:1) at concentrations of 20, 30, 40, 50, 55, 60, 65 and 70 µg/mL and 20, 30, 40, 50, 55, 60, 65, 70 and 80 µg/mL in the absence and presence of mammalian metabolic activation (S9-mix), respectively.
The test item was tested up to cytotoxic and solubility limits. The positive controls did induce the appropriate response. There was no evidence of induced mutant colonies over background levels at the concentrations tested.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.
Referenceopen allclose all
Table 1 Raw plate counts and calculated mutagenicity data, experiment 1 (- S9 mix)
Strain |
Compound |
Conc. level (µg/plate) |
Mean revertants per plate |
SD |
Fold increase |
Dunnett’s T-value |
Statistic. sig. |
Revertant numbers/plate |
TA98 |
DMSO |
- |
22.0 |
1.7 |
- |
- |
- |
21, 24, 21 |
Test item |
5 |
20.0 |
1.0 |
0.9 |
-0.36 |
NS |
21, 19, 20 |
|
16 |
21.7 |
6.0 |
1.0 |
-0.10 |
NS |
28, 16, 21 |
||
50 |
14.3 |
4.7 |
0.7 |
-1.55 |
NS |
16, 9, 18 |
||
160 |
19.0 |
7.0 |
0.9 |
-0.62 |
NS |
27, 14, 16 |
||
500 |
15.0 |
10.4 |
0.7 |
-1.57 |
NS |
9, 9, 27 |
||
1600 |
2.0 |
1.7 |
0.1 |
-5.51 |
NS |
1S, 1MS, 4S |
||
5000 |
2.0 |
1.7 |
0.1 |
-5.81 |
NS |
3SP, 3SP, 0SP |
||
2NF |
5 |
419.0 |
97.3 |
19.0 |
- |
- |
527, 338, 392 |
|
TA100 |
DMSO |
- |
117.3 |
6.8 |
- |
- |
NS |
112, 115, 125 |
Test item |
5 |
96.0 |
21.0 |
0.8 |
-1.62 |
NS |
120, 87, 81 |
|
16 |
95.3 |
19.2 |
0.8 |
-1.66 |
NS |
116, 92, 78 |
||
50 |
84.3 |
7.8 |
0.7 |
-2.50 |
NS |
78, 93, 82 |
||
160 |
101.0 |
12.5 |
0.9 |
-1.20 |
NS |
97, 115, 91 |
||
500 |
60.0 |
14.9 |
0.5 |
-4.72 |
NS |
54S, 49S, 77S |
||
1600 |
2.0 |
1.7 |
0.0 |
-14.64 |
NS |
3V, 0V, 3V |
||
5000 |
- |
- |
- |
- |
- |
-PT, -PT, -PT |
||
NaN3 |
2 |
450.0 |
20.4 |
3.8 |
- |
- |
515, 554, 507 |
|
TA1535 |
DMSO |
- |
8.7 |
3.2 |
- |
- |
- |
10, 5, 11 |
Test item |
5 |
9.7 |
0.6 |
1.1 |
0.39 |
NS |
10, 10, 9 |
|
16 |
14.3 |
7.6 |
1.7 |
1.54 |
NS |
11, 23, 9 |
||
50 |
10.3 |
6.8 |
1.2 |
0.38 |
NS |
5, 18, 8 |
||
160 |
11.3 |
3.2 |
1.3 |
0.85 |
NS |
9, 10, 15 |
||
500 |
9.3 |
4.0 |
1.1 |
0.19 |
NS |
5, 10, 13 |
||
1600 |
1.7 |
1.2 |
0.2 |
-3.20 |
NS |
1S, 3S, 1S |
||
5000 |
3.3 |
0.6 |
0.4 |
-2.09 |
NS |
4SP, 3SP, 3SP |
||
NaN3 |
2 |
525.3 |
25.1 |
60.6 |
- |
- |
515, 554, 507 |
|
TA1537 |
DMSO |
- |
6.0 |
2.6 |
- |
- |
- |
5, 9, 4 |
Test item |
5 |
7.0 |
2.6 |
1.2 |
0.31 |
NS |
4, 8, 9 |
|
16 |
9.0 |
7.5 |
1.5 |
0.49 |
NS |
1, 10, 6 |
||
50 |
5.0 |
2.6 |
0.8 |
-0.36 |
NS |
4, 3, 8 |
||
160 |
7.0 |
2.6 |
1.2 |
0.31 |
NS |
4, 9, 8 |
||
500 |
4.0 |
0.0 |
0.7 |
-0.65 |
NS |
4, 4, 4 |
||
1600 |
- |
- |
- |
- |
- |
-T, -T, -T |
||
5000 |
- |
- |
- |
- |
- |
-PT, -PT, -PT |
||
AAC |
50 |
218.3 |
109.5 |
36.4 |
- |
- |
326, 222, 107 |
|
TA102 |
DMSO |
- |
246.7 |
45.7 |
- |
- |
- |
299, 226, 215 |
Test item |
5 |
229.0 |
10.6 |
0.9 |
-0.78 |
NS |
237, 233, 217 |
|
16 |
257.0 |
41.6 |
1.0 |
0.49 |
NS |
231, 305, 235 |
||
50 |
233.3 |
11.0 |
0.9 |
-0.57 |
NS |
221, 237, 242 |
||
160 |
169.0 |
5.0 |
0.7 |
-3.88 |
NS |
164S, 174S, 169S |
||
500 |
58.3 |
8.5 |
0.2 |
-11.71 |
NS |
67S, 50S, 58S |
||
1600 |
- |
- |
- |
- |
- |
-T, -T, -T |
||
5000 |
- |
- |
- |
- |
- |
-PT, -PT, -PT |
||
MMC |
0.2 |
696.3 |
174.4 |
2.8 |
- |
- |
657, 545,887 |
P: precipitation of test item observed
S: slight thinning of background bacterial lawn
T: toxic, no revertant colonies
V: very thin background bacterial lawn
Table 2 Raw plate counts and calculated mutagenicity data, experiment 1 (+ S9 mix)
Strain |
Compound |
Conc. level (µg/plate) |
Mean revertants per plate |
SD |
Fold increase |
Dunnett’s T-value |
Statistic. sig. |
Revertant numbers/plate |
TA98 |
DMSO |
- |
26.7 |
2.5 |
- |
- |
- |
24, 29, 27 |
Test item |
5 |
26.7 |
6.4 |
1.0 |
-0.04 |
NS |
34, 23, 23 |
|
16 |
17.7 |
3.2 |
0.7 |
-1.78 |
NS |
20, 19, 14 |
||
50 |
20.3 |
4.0 |
0.8 |
-1.22 |
NS |
25, 18, 18 |
||
160 |
27.0 |
10.1 |
1.0 |
-0.04 |
NS |
25, 18, 38 |
||
500 |
12.7 |
3.2 |
0.5 |
-2.98 |
NS |
9, 14, 15 |
||
1600 |
8.0 |
2.0 |
0.3 |
-4.31 |
NS |
8S, 10S, 6S |
||
5000 |
3.0 |
2.6 |
0.1 |
-6.88 |
NS |
0SP, 4SP, 5SP |
||
B[a]P |
10 |
296.0 |
81.7 |
11.1 |
- |
- |
324, 360, 204 |
|
TA100 |
DMSO |
- |
111.0 |
21.8 |
- |
- |
- |
136, 96, 101 |
Test item |
5 |
113.0 |
11.1 |
1.0 |
0.21 |
NS |
103, 125, 111 |
|
16 |
118.0 |
18.4 |
1.1 |
0.59 |
NS |
126, 97, 131 |
||
50 |
102.0 |
19.3 |
0.9 |
-0.76 |
NS |
80, 116, 110 |
||
160 |
110.3 |
8.4 |
1.0 |
-0.01 |
NS |
120, 106, 105 |
||
500 |
65.7 |
7.2 |
0.6 |
-4.20 |
NS |
74S, 61S, 62S |
||
1600 |
4.3 |
1.2 |
0.0 |
-14.73 |
NS |
5V, 5V, 3V |
||
5000 |
- |
- |
- |
- |
- |
-PT, -PT, -PT |
||
AAN |
5 |
1045.7 |
47.5 |
9.4 |
- |
- |
1030, 1008, 1099 |
|
TA1535 |
DMSO |
- |
18.0 |
5.3 |
- |
- |
- |
14, 16, 24 |
Test item |
5 |
12.3 |
3.2 |
0.7 |
-1.32 |
NS |
16, 10, 11 |
|
16 |
10.7 |
8.3 |
0.6 |
-2.04 |
NS |
20, 4, 8 |
||
50 |
12.7 |
6.8 |
0.7 |
-1.39 |
NS |
5, 18, 15 |
||
160 |
9.7 |
1.5 |
0.5 |
-2.03 |
NS |
10, 11, 8 |
||
500 |
13.7 |
2.5 |
0.8 |
-0.96 |
NS |
16, 14, 11 |
||
1600 |
5.0 |
1.0 |
0.3 |
-3.63 |
NS |
6S, 5S, 4S |
||
5000 |
4.7 |
1.2 |
0.3 |
-3.77 |
NS |
6SP, 4SP, 4MSP |
||
AAN |
5 |
183.3 |
46.2 |
10.2 |
- |
- |
130, 212, 208 |
|
TA1537 |
DMSO |
- |
8.0 |
2.6 |
- |
- |
- |
5, 10, 9 |
Test item |
5 |
10.7 |
0.6 |
1.3 |
1.02 |
NS |
11, 11, 10 |
|
16 |
9.0 |
2.6 |
1.1 |
0.39 |
NS |
11, 6, 10 |
||
50 |
9.0 |
4.6 |
1.1 |
0.30 |
NS |
8, 5, 14 |
||
160 |
8.0 |
2.0 |
1.0 |
0.03 |
NS |
8, 10, 6 |
||
500 |
4.7 |
0.6 |
0.6 |
-1.41 |
NS |
4, 5, 5 |
||
1600 |
2.0 |
1.7 |
0.3 |
-3.60 |
NS |
0S, 3S, 3S |
||
5000 |
- |
- |
- |
- |
- |
-PT, -PT, -PT |
||
AAN |
|
245.7 |
45.7 |
30.7 |
- |
- |
196, 286, 255 |
|
TA102 |
DMSO |
- |
205.3 |
23.4 |
- |
- |
- |
188, 196, 232 |
Test item |
5 |
222.3 |
22.5 |
1.1 |
0.85 |
NS |
230, 240, 197 |
|
16 |
218.3 |
14.6 |
1.1 |
0.67 |
NS |
223, 202, 230 |
||
50 |
245.7 |
1.5 |
1.2 |
1.98 |
NS |
243, 244, 247 |
||
160 |
228.3 |
44.2 |
1.1 |
1.09 |
NS |
187,223, 275 |
||
500 |
132.0 |
18.1 |
0.6 |
-4.15 |
NS |
151S, 130S, 115S |
||
1600 |
- |
- |
- |
- |
- |
-T, -T, -T |
||
5000 |
- |
- |
- |
- |
- |
-PT, -PT, -PT |
||
AAN |
20 |
1697.3 |
111.4 |
8.3 |
- |
- |
1686, 1589, 1817 |
P: precipitation of test item observed
S: slight thinning of background bacterial lawn
T: toxic, no revertant colonies
V: very thin background bacterial lawn
Table 3 Raw plate counts and calculated mutagenicity data, experiment 2 (- S9 mix)
Strain |
Compound |
Conc. level (µg/plate) |
Mean revertants per plate |
SD |
Fold increase |
Dunnett’s T-value |
Statistic. sig. |
Revertant numbers/plate |
TA98 |
DMSO |
- |
22.0 |
7.2 |
- |
- |
- |
16, 30, 20 |
Test item |
50 |
23.3 |
8.0 |
1.1 |
0.24 |
NS |
24, 15, 31 |
|
100 |
23.7 |
8.5 |
1.1 |
0.29 |
NS |
24, 32, 15 |
||
200 |
15.7 |
5.0 |
0.7 |
-1.34 |
NS |
11, 15, 21 |
||
400 |
16.0 |
3.5 |
0.7 |
-1.22 |
NS |
14, 20, 14 |
||
800 |
11.0 |
1.0 |
0.5 |
-2.46 |
NS |
10S, 11S, 12S |
||
1600 |
2.7 |
2.1 |
0.1 |
-5.71 |
NS |
2S, 1S, 5S |
||
2NF |
5 |
323.7 |
60.7 |
14.7 |
- |
- |
310, 271, 390 |
|
TA100 |
DMSO |
- |
74.3 |
7.5 |
- |
- |
- |
82, 74, 67 |
Test item |
50 |
82.7 |
5.1 |
1.1 |
1.10 |
NS |
87, 77, 84 |
|
100 |
84.3 |
5.0 |
1.1 |
1.31 |
NS |
85, 89, 79 |
||
200 |
76.7 |
10.5 |
1.0 |
0.30 |
NS |
87, 77, 66 |
||
400 |
43.7 |
4.0 |
0.6 |
-4.66 |
NS |
46S, 39S, 46S |
||
800 |
13.7 |
7.2 |
0.2 |
-11.58 |
NS |
22S, 9S, 10S |
||
1600 |
- |
- |
- |
- |
- |
-T, -T, -T |
||
NaN3 |
2 |
415.0 |
1.7 |
5.6 |
- |
- |
414, 414, 417 |
|
TA1535 |
DMSO |
- |
15.3 |
3.5 |
- |
- |
- |
19, 15, 12 |
Test item |
50 |
12.0 |
3.6 |
0.8 |
-1.34 |
NS |
16, 9, 11 |
|
100 |
12.7 |
2.1 |
0.8 |
-1.02 |
NS |
15, 11, 12 |
||
200 |
11.0 |
0.0 |
0.7 |
-1.70 |
NS |
11, 11, 11 |
||
400 |
15.0 |
4.0 |
1.0 |
-0.14 |
NS |
15, 19, 11 |
||
800 |
7.3 |
1.5 |
0.5 |
-3.51 |
NS |
9S, 7S, 6S |
||
1600 |
2.0 |
1.7 |
0.1 |
-7.50 |
NS |
1MSB, 1S, 4S |
||
NaN3 |
2 |
428.3 |
25.0 |
27.9 |
- |
- |
434, 450, 401 |
|
TA1537 |
DMSO |
- |
14.3 |
4.9 |
- |
- |
- |
20, 11, 12 |
Test item |
50 |
10.7 |
5.0 |
0.7 |
-0.76 |
NS |
16, 10, 6 |
|
100 |
12.0 |
8.5 |
0.8 |
-0.62 |
NS |
21, 4, 11 |
||
200 |
11.0 |
5.0 |
0.8 |
-0.69 |
NS |
6, 16, 11 |
||
400 |
5.7 |
3.5 |
0.4 |
-2.02 |
NS |
9S, 2S, 6S |
||
800 |
- |
- |
- |
- |
- |
-T, -T, -T |
||
1600 |
- |
- |
- |
- |
- |
-T, -T, -T |
||
AAC |
50 |
86.7 |
32.1 |
6.0 |
- |
- |
100, 50, 110 |
|
TA102 |
DMSO |
- |
242.0 |
26.5 |
- |
- |
- |
271, 236, 219 |
Test item |
50 |
229.3 |
28.1 |
0.9 |
-0.25 |
NS |
256, 232, 200 |
|
100 |
142.7 |
87.2 |
0.6 |
-2.51 |
NS |
42, 195, 191 |
||
200 |
164.7 |
10.8 |
0.7 |
-1.65 |
NS |
160S, 157S, 177S |
||
400 |
105.7 |
9.1 |
0.4 |
-3.20 |
NS |
102S, 99S, 116S |
||
800 |
- |
- |
- |
- |
- |
-T, -T, -T |
||
1600 |
- |
- |
- |
- |
- |
-T, -T, -T |
||
MMC |
0.2 |
594.7 |
30.7 |
2.5 |
- |
- |
579, 575, 630 |
P: precipitation of test item observed
S: slight thinning of background bacterial lawn
T: toxic, no revertant colonies
V: very thin background bacterial lawn
Table 4 Raw plate counts and calculated mutagenicity data, experiment 2 (+ S9 mix)
Strain |
Compound |
Conc. level (µg/plate) |
Mean revertants per plate |
SD |
Fold increase |
Dunnett’s T-value |
Statistic. sig. |
Revertant numbers/plate |
TA98 |
DMSO |
- |
27.7 |
6.7 |
- |
- |
- |
20, 32, 31 |
Test item |
50 |
23.3 |
5.5 |
0.8 |
-0.92 |
NS |
27, 26, 17 |
|
100 |
35.7 |
3.5 |
1.3 |
1.59 |
NS |
32, 36, 39 |
||
200 |
30.0 |
10.5 |
1.1 |
0.41 |
NS |
29, 20, 41 |
||
400 |
27.3 |
2.9 |
1.0 |
-0.02 |
NS |
29S, 24S, 29S |
||
800 |
14.0 |
3.0 |
0.5 |
-3.25 |
NS |
14S, 11S, 17S |
||
1600 |
12.3 |
3.2 |
0.4 |
-3.76 |
NS |
10S, 16S, 11S |
||
B[a]P |
10 |
304.3 |
44.3 |
11.0 |
- |
- |
354, 290, 269 |
|
TA100 |
DMSO |
- |
115.7 |
21.8 |
- |
- |
- |
92, 120, 135 |
Test item |
50 |
113.7 |
4.2 |
1.0 |
-0.09 |
NS |
117, 109, 115 |
|
100 |
104.0 |
5.3 |
0.9 |
-0.80 |
NS |
102, 100, 110 |
||
200 |
107.0 |
24.9 |
0.9 |
-0.65 |
NS |
134, 102, 85 |
||
400 |
53.0 |
10.4 |
0.5 |
-5.30 |
NS |
59S, 41S, 59S |
||
800 |
24.0 |
8.7 |
0.2 |
-8.97 |
NS |
34S, 19S, 19S |
||
1600 |
- |
- |
- |
- |
- |
-T, -T, -T |
||
AAN |
5 |
688.0 |
77.4 |
5.9 |
- |
- |
774, 666, 624 |
|
TA1535 |
DMSO |
- |
15.3 |
0.6 |
- |
- |
- |
15, 16, 15 |
Test item |
50 |
20.0 |
5.2 |
1.3 |
1.83 |
NS |
17, 17, 26 |
|
100 |
17.0 |
3.0 |
1.1 |
0.68 |
NS |
20, 14, 17 |
||
200 |
14.7 |
3.2 |
1.0 |
-0.35 |
NS |
16, 17, 11 |
||
400 |
15.0 |
1.0 |
1.0 |
-0.15 |
NS |
14S, 15S, 16S |
||
800 |
8.3 |
2.3 |
0.5 |
-3.59 |
NS |
7S, 7S, 11S |
||
1600 |
7.3 |
1.5 |
0.5 |
-4.18 |
NS |
6V, 9V, 7V |
||
AAN |
5 |
167.7 |
11.0 |
10.9 |
- |
- |
179, 157, 167 |
|
TA1537 |
DMSO |
- |
11.7 |
2.1 |
- |
- |
- |
11, 10, 14 |
Test item |
50 |
11.7 |
4.5 |
1.0 |
-0.09 |
NS |
12, 7, 16 |
|
100 |
14.0 |
5.2 |
1.2 |
0.70 |
NS |
11, 11, 20 |
||
200 |
11.3 |
2.5 |
1.0 |
-0.13 |
NS |
11, 14, 9 |
||
400 |
12.7 |
3.1 |
1.1 |
0.32 |
NS |
12S, 16S, 10S |
||
800 |
- |
- |
- |
- |
- |
-T, -T, -T |
||
1600 |
- |
- |
- |
- |
- |
-T, -T, -T |
||
AAN |
5 |
219.3 |
82.9 |
18.8 |
- |
- |
169, 174, 315 |
|
TA102 |
DMSO |
- |
277.3 |
19.1 |
- |
- |
- |
257, 280, 295 |
Test item |
50 |
262.0 |
2.0 |
0.9 |
-0.97 |
NS |
262, 264, 260 |
|
100 |
266.7 |
10.7 |
1.0 |
-0.67 |
NS |
260, 279, 261 |
||
200 |
253.0 |
25.6 |
0.9 |
-1.58 |
NS |
226, 277, 256 |
||
400 |
199.3 |
19.4 |
0.7 |
-5.33 |
NS |
209S, 212S, 177S |
||
800 |
107.0 |
14.1 |
0.4 |
-13.25 |
NS |
105S, 122S, 94S |
||
1600 |
- |
- |
- |
- |
- |
-T, -T, -T |
||
AAN |
20 |
1826.3 |
118.6 |
6.6 |
- |
- |
1711, 1820, 1948 |
P: precipitation of test item observed
S: slight thinning of background bacterial lawn
T: toxic, no revertant colonies
V: very thin background bacterial lawn
Table 1 3 hour treatment in the absence of S9 with 17 hour recovery (3+17),– range finder
Treatment (µg/mL) |
Replicate |
Mitotic Index (%) |
MIH* (%) |
Vehicle |
A |
5.0 |
|
B |
4.3 |
|
|
Total |
47 |
- |
|
4.354 |
A |
4.4 |
5 |
7.256 |
A |
3.8 |
18 |
12.09 |
A |
5.1 |
0 |
20.16 |
A |
4.7 |
0 |
33.59 |
A |
4.5 |
3 |
55.99 |
A |
3.6 |
23 |
93.31 |
A |
3.6 |
23 |
155.5 |
A |
0.1 |
98P |
259.2 |
A |
T |
-P |
432.0 |
A |
T |
-P |
720.0 |
A |
T |
-P |
1200 |
A |
T |
-P |
T: toxic
P: precipitation of test item at start of experiment
H: precipitation of test item observed at harvest
* mitotic inhibition
Table 2 3 hour treatment in the presence of S9 with 17 hour recovery (3+17),– range finder
Treatment (µg/mL) |
Replicate |
Mitotic Index (%) |
MIH* (%) |
Vehicle |
A |
5.2 |
|
B |
6.6 |
|
|
Total |
5.9 |
- |
|
4.354 |
A |
5.6 |
5 |
7.256 |
A |
5.4 |
8 |
12.09 |
A |
5.5 |
7 |
20.16 |
A |
3.6 |
39 |
33.59 |
A |
5.7 |
3 |
55.99 |
A |
4.7 |
20 |
93.31 |
A |
3.9 |
34 |
155.5 |
A |
0.1 |
98P |
259.2 |
A |
T |
-P |
432.0 |
A |
T |
-P |
720.0 |
A |
T |
-PH |
1200 |
A |
T |
-PH |
T: toxic
P: precipitation of test item at start of experiment
H: precipitation of test item observed at harvest
* mitotic inhibition
Table 3 20 hour treatment in the absence of S9 with 0 hour recovery (20+0),– range finder
Treatment (µg/mL) |
Replicate |
Mitotic Index (%) |
MIH* (%) |
Vehicle |
A |
6.1 |
|
B |
8.1 |
|
|
Total |
7.1 |
- |
|
4.354 |
A |
6.5 |
8 |
7.256 |
A |
6.9 |
3 |
12.09 |
A |
5.1 |
28 |
20.16 |
A |
4.4 |
38 |
33.59 |
A |
3.1 |
56 |
55.99 |
A |
2.5 |
65 |
93.31 |
A |
0.2 |
97 |
155.5 |
A |
0.0 |
-P |
259.2 |
A |
T |
-P |
432.0 |
A |
T |
-PH |
720.0 |
A |
T |
-PH |
1200 |
A |
T |
-PH |
T: toxic
P: precipitation of test item at start of experiment
H: precipitation of test item observed at harvest
* mitotic inhibition
No marked changes in osmolality or pH were observed at the highest concentration tested in the Range-Finder (1200 μg/mL), compared to the concurrent vehicle controls (individual data not reported).
The results of the cytotoxicity Range-Finder Experiment were used to select suitable concentrations for the Chromosome Aberration Experiment. Marked cytotoxicity (>60 % MIH) was observed at 155.5 μg/mL and above for the 3+17 treatments in the absence and presence of S-9 and at 55.99 μg/mL and above for the 20+0 hour treatment in the absence of S-9. Based on these observations and in order to permit a range of cytotoxicity under each treatment condition, the maximum concentrations selected for the Chromosome Aberration Experiment were 175 μg/mL for the 3+17 hour treatments in the absence and presence of S-9 and 100 μg/mL for the 20+0 hour treatment in the absence of S-9.
The results of the MI determinations from the Chromosome Aberration Experiment were as follows:
Table 4 3 hour treatment in the absence of S9 with 17 hour recovery (3+17), chromosome aberration experiment
Treatment (µg/mL) |
Replicate |
Mitotic Index (%) |
Cytotoxicity based on MI* (%) |
Vehicle |
A |
10.8 |
|
B |
8.6 |
||
C |
8.1 |
||
D |
8.4 |
||
Total |
9.0 |
- |
|
10 |
A |
9.6 |
|
B |
8.2 |
||
Total |
8.9 |
1 |
|
20 |
A |
8.4 |
|
B |
8.5 |
||
Total |
8.5 |
6 |
|
40 |
A |
8.0 |
|
B |
8.5 |
||
Total |
8.3 |
8# |
|
60 |
A |
7.0 |
|
B |
7.5 |
||
Total |
7.3 |
19 |
|
80 |
A |
6.5 |
|
B |
6.2 |
||
Total |
6.4 |
29# |
|
90 |
A |
6.4 |
|
B |
5.2 |
||
Total |
5.8 |
35 |
|
100 |
A |
5.6 |
|
B |
5.4 |
||
Total |
5.5 |
39 |
|
110 |
A |
4.7 |
|
B |
4.7 |
||
Total |
4.7 |
48 |
|
120 |
A |
3.7 |
|
B |
3.9 |
||
Total |
3.8 |
58# |
|
130 |
A |
2.5 |
|
B |
3.6 |
||
Total |
3.1 |
66 |
|
140 |
A |
3.2 |
|
B |
3.0 |
||
Total |
3.1 |
65P |
|
150 |
A |
1.3 |
|
B |
1.3 |
||
Total |
1.3 |
86P |
|
175 |
A |
T |
|
B |
T |
||
Total |
- |
-P |
|
MMC, 0.3 |
A |
5.9 |
|
B |
5.2 |
||
Total |
5.6 |
38# |
|
MMC, 0.4 |
A |
3.4 |
|
B |
3.5 |
||
Total |
3.5 |
62 |
T: toxic
P: precipitation of test item at the start of the experiment
# concentrations selected for chromosome aberration analysis
* mitotic inhibition
Table 5 3 hour treatment in the presence of S9 with 17 hour recovery (3+17), chromosome aberration experiment
Treatment (µg/mL) |
Replicate |
Mitotic Index (%) |
Cytotoxicity based on MI* (%) |
Vehicle |
A |
9.7 |
|
B |
7.7 |
||
C |
10.4 |
||
D |
10.5 |
||
Total |
9.6 |
- |
|
10 |
A |
9.1 |
|
B |
9.9 |
||
Total |
9.5 |
1 |
|
20 |
A |
9.7 |
|
B |
11.2 |
||
Total |
10.5 |
0# |
|
40 |
A |
10.6 |
|
B |
6.9 |
||
Total |
8.8 |
9 |
|
60 |
A |
7.7 |
|
B |
7.4 |
||
Total |
7.6 |
21 |
|
80 |
A |
7.7 |
|
B |
6.8 |
||
Total |
7.3 |
24# |
|
90 |
A |
NM |
|
B |
NM |
||
Total |
- |
- |
|
100 |
A |
5.7 |
|
B |
6.4 |
||
Total |
6.1 |
37 |
|
110 |
A |
4.8 |
|
B |
4.4 |
||
Total |
4.6 |
52# |
|
120 |
A |
3.0 |
|
B |
5.7 |
||
Total |
4.4 |
55 |
|
130 |
A |
2.6 |
|
B |
3.6 |
||
Total |
3.1 |
68 |
|
140 |
A |
1.9 |
|
B |
1.9 |
||
Total |
1.9 |
80 |
|
150 |
A |
0.3 |
|
B |
0.5 |
||
Total |
0.4 |
96 |
|
175 |
A |
T |
|
B |
T |
||
Total |
- |
- |
|
CPA, 1 |
A |
9.2 |
|
B |
10.1 |
||
Total |
9.7 |
0 |
|
CPA, 2 |
A |
5.8 |
|
B |
4.4 |
||
Total |
5.1 |
47# |
T: toxic
NM: slides not made due to mechanical error
# concentrations selected for chromosome aberration analysis
* mitotic inhibition
Table 6 20 hour treatment in the absence of S9 with 0 hour recovery (20+0), chromosome aberration experiment
Treatment (µg/mL) |
Replicate |
Mitotic Index (%) |
Cytotoxicity based on MI* (%) |
Vehicle |
A |
6.4 |
|
B |
7.4 |
||
C |
6.3 |
||
D |
6.1 |
||
Total |
6.6 |
- |
|
5 |
A |
6.6 |
|
B |
6.5 |
||
Total |
6.6 |
0 |
|
10 |
A |
5.9 |
|
B |
6.5 |
||
Total |
6.2 |
5 |
|
15 |
A |
6.5 |
|
B |
5.0 |
||
Total |
5.8 |
12# |
|
20 |
A |
4.7 |
|
Total |
5.4 |
18 |
|
25 |
A |
4.4 |
|
B |
5.3 |
||
Total |
4.9 |
26 |
|
30 |
A |
4.5 |
|
B |
3.7 |
||
Total |
4.1 |
37# |
|
35 |
A |
3.4 |
|
B |
3.3 |
||
Total |
3.4 |
49 |
|
40 |
A |
2.9 |
|
B |
3.2 |
||
Total |
3.1 |
53# |
|
45 |
A |
1.9 |
|
B |
2.8 |
||
Total |
2.4 |
64 |
|
50 |
A |
2.0 |
|
B |
1.8 |
||
Total |
1.9 |
71 |
|
60 |
A |
2.0 |
|
B |
0.9 |
||
Total |
1.5 |
78 |
|
80 |
A |
0.7 |
|
B |
0.4 |
||
Total |
0.6 |
92 |
|
100 |
A |
0.3 |
|
B |
0.5 |
||
Total |
0.4 |
94 |
|
MMC 0.05 |
A |
4.5 |
|
B |
5.1 |
||
Total |
4.8 |
27 |
|
MMC 0.1 |
A |
3.6 |
|
B |
3.6 |
||
Total |
3.6 |
45# |
# concentrations selected for chromosome aberration analysis
* mitotic inhibition
Study validity
1. The binomial dispersion test demonstrated acceptable heterogeneity between replicate cultures.
2. The proportions of cells with structural aberrations (excluding gaps) in vehicle control cultures fell within the normal ranges.
3. At least 300 cells were suitable for analysis at each concentration, unless 15 or more cells showing structural aberrations (per slide) other than gaps only were observed during analysis.
4. The positive control chemicals induced statistically significant increases in the proportion of cells with structural aberrations. Both replicate cultures at the positive control concentration analysed under each treatment condition demonstrated structural aberration cell frequencies (excluding gaps) that clearly exceeded the current historical vehicle control ranges
5. The maximum concentration analysed under each treatment condition was a concentration inducing approximately 50% cytotoxicity.
Structural aberrations
Treatment of cultures with the test article for 3+17 hours in the absence of S-9 resulted in frequencies of cells with structural chromosome aberrations that were significantly higher (p ≤ 0.05), compared to the concurrent vehicle controls, at the lowest concentration analysed (40 μg/mL). However, the aberration frequencies (excluding gaps) were within the normal range at all concentrations analysed and the statistical significance was exacerbated by a chromosome aberration frequency of zero in both vehicle control cultures.
Treatment of cultures for 3+17 hours in the presence of S-9 resulted in frequencies of cells with structural chromosome aberrations that were similar to and not statistically higher (at the p ≤ 0.05 level) than the concurrent vehicle controls at all concentrations analysed. The aberration frequencies (excluding gaps) fell within the normal range at all concentrations analysed.
Treatment of cultures for 20+0 hours in the absence of S-9 resulted in frequencies of cells with structural chromosome aberrations that were generally similar to and not significantly higher (at the p ≤ 0.05 level) than the concurrent vehicle controlsat all concentrations analysed. The aberration frequency (excluding gaps) marginally exceeded the normal range in one culture at the highest concentration analysed (40 μg/mL) but the aberration frequency in the replicate culture was zero, therefore this isolated increase was considered not biologically relevant.
Table 7 3 hour treatment in the absence of S9 with 17 hour recovery (3+17), chromosome aberration experiment – numbers and types of structural aberrations observed
Treatment (µg/mL) |
Replicate |
Cells scored |
Cells with aberrations inc. gaps |
Cells with aberrations exc. gaps |
Significance§ |
MIH* (%) |
Vehicle |
A |
150 |
0 |
0 |
|
|
B |
150 |
0 |
0 |
|||
Total |
300 |
0 (0.00 %) |
0 (0.00 %) |
- |
- |
|
40 |
A |
150 |
3 |
3 |
|
|
B |
150 |
1 |
1 |
|||
Total |
300 |
4 (1.33 %) |
4 (1.33 %) |
p ≤ 0.05 |
8 |
|
80 |
A |
150 |
0 |
0 |
|
|
B |
150 |
3 |
2 |
|||
Total |
300 |
3 (1.00 %) |
2 (0.67 %) |
NS |
29 |
|
120 |
A |
150 |
0 |
0 |
|
|
B |
150 |
2 |
1 |
|||
Total |
300 |
2 (0.67 %) |
1 (0.33 %) |
NS |
58 |
|
MMC 0.3 |
A |
145 |
23# |
22# |
|
|
B |
136 |
28# |
26# |
|||
Total |
281 |
51 (18.15 %) |
48 (17.08 %) |
p ≤ 0.001 |
38 |
NS: not significant
§statistical significance
# numbers exceed historical vehicle control range
* mitotic inhibition
Table 8 3 hour treatment in the presence of S9 with 17 hour recovery (3+17), chromosome aberration experiment – cells with structural aberrations
Treatment (µg/mL) |
Replicate |
Cells scored |
Cells with aberrations inc. gaps |
Cells with aberrations exc. gaps |
Significance§ |
MIH* (%) |
Vehicle |
A |
150 |
0 |
0 |
|
|
B |
150 |
2 |
1 |
|||
Total |
300 |
2 (0.67 %) |
1 (0.33 %) |
- |
- |
|
20 |
A |
150 |
0 |
0 |
|
|
B |
150 |
2 |
1 |
|||
Total |
300 |
2 (0.67 %) |
1 (0.33 %) |
NS |
0 |
|
80 |
A |
150 |
0 |
0 |
|
|
B |
150 |
2 |
2 |
|||
Total |
300 |
2 (0.67 %) |
2 (0.67 %) |
NS |
24 |
|
110 |
A |
150 |
1 |
1 |
|
|
B |
150 |
1 |
0 |
|||
Total |
300 |
2 (0.67 %) |
1 (0.33 %) |
NS |
52 |
|
CPA 2 |
A |
147 |
32# |
29# |
|
|
B |
136 |
25# |
24# |
|||
Total |
283 |
57 (20.14 %) |
53 (18.73 %) |
p ≤ 0.001 |
47 |
NS: not significant
§statistical significance
# numbers exceed historical vehicle control range
* mitotic inhibition
Table 9 20 hour treatment in the absence of S9 with 0 hour recovery (20+0), chromosome aberration experiment – cells with structural aberrations
Treatment (µg/mL) |
Replicate |
Cells scored |
Cells with aberrations inc. gaps |
Cells with aberrations exc. gaps |
Significance§ |
MIH* (%) |
Vehicle |
A |
150 |
0 |
0 |
|
|
B |
150 |
1 |
1 |
|||
Total |
300 |
1 (0.33 %) |
1 (0.33 %) |
- |
- |
|
15 |
A |
150 |
2 |
1 |
|
|
B |
150 |
1 |
1 |
|||
Total |
300 |
3 (1.00 %) |
2 (0.67 %) |
NS |
12 |
|
30 |
A |
150 |
0 |
0 |
|
|
B |
150 |
1 |
0 |
|||
Total |
300 |
1 (0.33 %) |
0 (0.00 %) |
NS |
37 |
|
40 |
A |
150 |
5# |
5# |
|
|
B |
150 |
0 |
0 |
|||
Total |
300 |
5 (1.67 %) |
5 (1.67 %) |
NS |
53 |
|
MMC 0.1 |
A |
144 |
24# |
23# |
|
|
B |
106 |
31# |
29# |
|||
Total |
250 |
55 (22.00 %) |
52 (20.80 %) |
p ≤ 0.001 |
45 |
NS: not significant
§statistical significance
# numbers exceed historical vehicle control range
* mitotic inhibition
Table 10 3 hour treatment in the absence of S9 with 17 hour recovery (3+17), chromosome aberration experiment – numbers and types of structural aberrations observed
Treatment (µg/mL) |
Rep |
Cells* |
G |
Chr del. |
Chr exch. |
Ctd del. |
Ctd exch. |
Other |
Abs +g |
Abs -g |
Vehicle |
A |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Total |
300 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
40.0 |
A |
150 |
0 |
0 |
0 |
3 |
0 |
0 |
3 |
3 |
B |
150 |
0 |
0 |
0 |
1 |
0 |
0 |
1 |
1 |
|
Total |
300 |
0 |
0 |
0 |
4 |
0 |
0 |
4 |
4 |
|
80.0 |
A |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
150 |
1 |
1 |
0 |
1 |
0 |
0 |
3 |
2 |
|
Total |
300 |
1 |
1 |
0 |
1 |
0 |
0 |
3 |
2 |
|
120.0 |
A |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
150 |
1 |
0 |
0 |
1 |
0 |
0 |
2 |
1 |
|
Total |
300 |
1 |
0 |
0 |
1 |
0 |
0 |
2 |
1 |
|
MMC 0.3 |
A |
145 |
3 |
0 |
0 |
21 |
6 |
0 |
30 |
27 |
B |
136 |
5 |
4 |
0 |
27 |
9 |
1 |
46 |
41 |
|
Total |
281 |
8 |
4 |
0 |
48 |
15 |
1 |
76 |
68 |
* total cells examined for structural aberrations
G: gaps
Chr del: chromosome deletion
Chr exch: chromosome exchanges
Ctd del: chromatid deletion
Ctd exch: chromatid exchanges
Abs: aberrations
Table 11 3 hour treatment in the absence of S9 with 17 hour recovery (3+17), chromosome aberration experiment – numbers and types of structural aberrations observed
Treatment (µg/mL) |
Rep |
Cells* |
G |
Chr del. |
Chr exch. |
Ctd del. |
Ctd exch. |
Other |
Abs +g |
Abs -g |
Vehicle |
A |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
150 |
1 |
0 |
0 |
1 |
0 |
0 |
2 |
1 |
|
Total |
300 |
1 |
0 |
0 |
1 |
0 |
0 |
2 |
1 |
|
40.0 |
A |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
150 |
1 |
1 |
0 |
0 |
0 |
0 |
2 |
1 |
|
Total |
300 |
1 |
1 |
0 |
0 |
0 |
0 |
2 |
1 |
|
80.0 |
A |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
150 |
0 |
2 |
0 |
0 |
0 |
0 |
2 |
2 |
|
Total |
300 |
0 |
2 |
0 |
0 |
0 |
0 |
2 |
2 |
|
120.0 |
A |
150 |
0 |
0 |
0 |
1 |
0 |
0 |
1 |
1 |
B |
150 |
1 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
|
Total |
300 |
1 |
0 |
0 |
1 |
0 |
0 |
2 |
1 |
|
MMC 0.3 |
A |
147 |
3 |
7 |
0 |
21 |
7 |
0 |
38 |
35 |
B |
136 |
1 |
1 |
0 |
34 |
3 |
0 |
39 |
38 |
|
Total |
283 |
4 |
8 |
0 |
55 |
10 |
0 |
77 |
73 |
* total cells examined for structural aberrations
G: gaps
Chr del: chromosome deletion
Chr exch: chromosome exchanges
Ctd del: chromatid deletion
Ctd exch: chromatid exchanges
Abs: aberrations
Table 12 3 hour treatment in the presence of S9 with 17 hour recovery (3+17), chromosome aberration experiment – numbers and types of structural aberrations observed
Treatment (µg/mL) |
Rep |
Cells* |
G |
Chr del. |
Chr exch. |
Ctd del. |
Ctd exch. |
Other |
Abs +g |
Abs -g |
Vehicle |
A |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
150 |
0 |
0 |
0 |
1 |
0 |
0 |
1 |
1 |
|
Total |
300 |
0 |
0 |
0 |
1 |
0 |
0 |
1 |
1 |
|
15.0 |
A |
150 |
1 |
0 |
0 |
1 |
0 |
0 |
2 |
1 |
B |
150 |
0 |
0 |
0 |
1 |
0 |
0 |
1 |
1 |
|
Total |
300 |
1 |
0 |
0 |
2 |
0 |
0 |
3 |
2 |
|
30.0 |
A |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
150 |
1 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
|
Total |
300 |
1 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
|
40.0 |
A |
150 |
1 |
1 |
0 |
4 |
0 |
0 |
6 |
5 |
B |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Total |
300 |
1 |
1 |
0 |
4 |
0 |
0 |
6 |
5 |
|
MMC 0.1 |
A |
144 |
3 |
4 |
0 |
27 |
1 |
0 |
35 |
32 |
B |
106 |
3 |
5 |
0 |
22 |
8 |
0 |
38 |
35 |
|
Total |
250 |
6 |
9 |
0 |
49 |
9 |
0 |
73 |
67 |
* total cells examined for structural aberrations
G: gaps
Chr del: chromosome deletion
Chr exch: chromosome exchanges
Ctd del: chromatid deletion
Ctd exch: chromatid exchanges
Abs: aberrations
Numerical Aberrations
Small, sporadic increases in the frequencies of cells with numerical aberrations, due to endoreduplication or polyploidy, which marginally exceeded the concurrent controls and the normal ranges, were observed under all three treatment conditions. There were no consistent concentration related responses across the three treatment conditions but numerical aberrations were not analysed quantitatively, as this assay is not specifically designed for quantitative evaluation of polyploidy/aneugenicity.
Table 13 3 hour treatment in the absence of S9 with 17 hour recovery (3+17), chromosome aberration experiment – numbers and types of numerical aberrations observed
Treatment (µg/mL) |
Rep |
Cells* |
E |
P |
Total abs. |
% with numerical abs. |
Vehicle |
A |
150 |
0 |
0 |
0 |
0.0 |
B |
151 |
0 |
1 |
1 |
0.7 |
|
Total |
301 |
0 |
1 |
1 |
0.3 |
|
40.0 |
A |
151 |
0 |
1 |
1 |
0.7 |
B |
151 |
0 |
1 |
1 |
0.7 |
|
Total |
302 |
0 |
2 |
2 |
0.7 |
|
80.0 |
A |
152 |
0 |
2# |
2 |
1.3 |
B |
151 |
0 |
1 |
1 |
0.7 |
|
Total |
303 |
0 |
3 |
3 |
1.0 |
|
120.0 |
A |
155 |
0 |
5# |
5 |
3.2 |
B |
155 |
0 |
5# |
5 |
3.2 |
|
Total |
310 |
0 |
10 |
10 |
3.2 |
|
MMC 0.3 |
A |
145 |
0 |
0 |
0 |
0.0 |
B |
137 |
0 |
1 |
1 |
0.7 |
|
Total |
282 |
0 |
1 |
1 |
0.4 |
* total cells examined for numerical aberrations
# number exceeds historical vehicle control range
E: endoreduplicated
P: polyploid (> 68 chromosomes)
Table 14 3 hour treatment in the presence of S9 with 17 hour recovery (3+17), chromosome aberration experiment – numbers and types of numerical aberrations observed
Treatment (µg/mL) |
Rep |
Cells* |
E |
P |
Total abs. |
% with numerical abs. |
Vehicle |
A |
150 |
0 |
0 |
0 |
0.0 |
B |
150 |
0 |
0 |
0 |
0.0 |
|
Total |
300 |
0 |
0 |
0 |
0.0 |
|
20.0 |
A |
151 |
0 |
1 |
1 |
0.7 |
B |
152 |
0 |
2# |
2 |
1.3 |
|
Total |
303 |
0 |
3 |
3 |
1.0 |
|
80.0 |
A |
151 |
1# |
0 |
1 |
0.7 |
B |
151 |
0 |
1 |
1 |
0.7 |
|
Total |
302 |
1 |
1 |
2 |
0.7 |
|
110.0 |
A |
151 |
0 |
1 |
1 |
0.7 |
B |
157 |
0 |
7# |
7 |
4.5 |
|
Total |
308 |
0 |
8 |
8 |
2.6 |
|
CPA 2.0 |
A |
147 |
0 |
0 |
0 |
0.0 |
B |
136 |
0 |
0 |
0 |
0.0 |
|
Total |
283 |
0 |
0 |
0 |
0.0 |
* total cells examined for numerical aberrations
# number exceeds historical vehicle control range
E: endoreduplicated
P: polyploid (> 68 chromosomes)
Table 15 20 hour treatment in the absence of S9 with 0 hour recovery (20+0), chromosome aberration experiment – numbers and types of numerical aberrations observed
Treatment (µg/mL) |
Rep |
Cells* |
E |
P |
Total abs. |
% with numerical abs. |
Vehicle |
A |
150 |
0 |
0 |
0 |
0.0 |
B |
150 |
0 |
0 |
0 |
0.0 |
|
Total |
300 |
0 |
0 |
0 |
0.0 |
|
15.0 |
A |
151 |
0 |
1 |
1 |
0.7 |
B |
150 |
0 |
0 |
0 |
0.0 |
|
Total |
301 |
0 |
1 |
1 |
0.3 |
|
30.0 |
A |
150 |
0 |
0 |
0 |
0.0 |
B |
152 |
0 |
2# |
2 |
1.3 |
|
Total |
302 |
0 |
2 |
2 |
0.7 |
|
40.0 |
A |
151 |
0 |
1 |
1 |
0.7 |
B |
153 |
0 |
3# |
3 |
2.0 |
|
Total |
304 |
0 |
4 |
4 |
1.3 |
|
MMC 0.1 |
A |
144 |
0 |
0 |
0 |
0.0 |
B |
106 |
0 |
0 |
0 |
0.0 |
|
Total |
250 |
0 |
0 |
0 |
0.0 |
* total cells examined for numerical aberrations
# number exceeds historical vehicle control range
E: endoreduplicated
P: polyploid (> 68 chromosomes)
Table 16 3 hour treatment in the absence of S9 with 17 hour recovery (3+17), chromosome aberration experiment – statistical analysis
Treatment (µg/mL) |
Cells scored |
Aberrant cells |
Proportion |
Fisher’s exact test |
Significance |
Vehicle |
300 |
0 |
0.000 |
- |
- |
40 |
300 |
4 |
0.013 |
0.031 |
p ≤ 0.05 |
80 |
300 |
2 |
0.007 |
0.125 |
NS |
120 |
300 |
1 |
0.003 |
0.250 |
NS |
MMC 0.3 |
281 |
48 |
0.171 |
0.000 |
p ≤ 0.001 |
Binomial dispersion testc2: 4.030 DF: 4 p-value: 0.402 Significance: NS
Cochran-Armitage Linear Trend p-value: 0.433 Significance: NS
NS: not significant
DF: degrees of freedom
Table 17 3 hour treatment in the presence of S9 with 17 hour recovery (3+17), chromosome aberration experiment – statistical analysis
Treatment (µg/mL) |
Cells scored |
Aberrant cells |
Proportion |
Fisher’s exact test |
Significance |
Vehicle |
300 |
1 |
0.003 |
- |
- |
20 |
300 |
1 |
0.003 |
0.500 |
NS |
80 |
300 |
2 |
0.007 |
0.312 |
NS |
110 |
300 |
1 |
0.003 |
0.500 |
NS |
CPA 2.0 |
283 |
53 |
0.187 |
0.000 |
p ≤ 0.001 |
Binomial dispersion testc2: 5.023 DF: 4 p-value: 0.285 Significance: NS
Cochran-Armitage Linear Trend p-value: 0.421 Significance: NS
NS: not significant
DF: degrees of freedom
Table 18 20 hour treatment in the absence of S9 with 0 hour recovery (20+0), chromosome aberration experiment – statistical analysis
Treatment (µg/mL) |
Cells scored |
Aberrant cells |
Proportion |
Fisher’s exact test |
Significance |
Vehicle |
300 |
1 |
0.003 |
- |
- |
15 |
300 |
2 |
0.007 |
0.312 |
NS |
30 |
300 |
0 |
0.000 |
0.750 |
NS |
40 |
300 |
5 |
0.017 |
0.062 |
NS |
MMC 0.1 |
250 |
52 |
0.208 |
0.000 |
p ≤ 0.001 |
Binomial dispersion testc2: 6.088 DF: 4 p-value: 0.193 Significance: NS
Cochran-Armitage Linear Trend p-value: 0.056 Significance: NS
NS: not significant
DF: degrees of freedom
Toxicity
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 18.75 to 600 µg/mL (limited by precipitation of the formulated test article in culture medium). Upon addition of the test article to the cultures, precipitate was observed at the highest two concentrations tested in the absence and presence of S-9 (300 and 600 µg/mL).Following the 3 hour treatment incubation period, precipitate was observed at the highest concentration tested in the absence of S-9 (600 µg/mL) and at the highest two concentrations tested in the presence of S-9 (300 and 600 µg/mL). The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the presence of S-9 was retained and the higher concentration was discarded. All concentrations were retained in the absence of S-9. The two highest concentrations retained in the absence of S‑9 (300 and 600 µg/mL) and the highest concentration retained in the presence of S‑9 (300 µg/mL) were not plated for survival due to excessive toxicity. The highest concentration to give³10 % relative survival (RS) was 37.5 µg/mL, which gave 66 % and 62 % RS in the absence and presence of S-9, respectively (see Table 1):
Table 1 Range-Finder Experiment - 3 Hour Treatment in the Absence and Presence of S-9
Concentration |
3 Hour Treatment -S‑9 |
3 Hour Treatment +S‑9 |
µg/mL |
% RS |
% RS |
0 |
100 |
100 |
18.75 |
94 |
69 |
37.5 |
66 |
62 |
75 |
0 |
3 |
150 |
0 |
0 |
300 |
P, NP |
P, PP, NP |
600 |
P, PP, NP |
P, PP, NP |
% RS Percent Relative Survival
P Precipitation observed at time of treatment
PP Precipitation observed at end of treatment incubation period
NP Not plated
No marked changes in osmolality or pH were observed in the Range-Finder at the lowest precipitating concentrations analysed in the absence and presence of S-9 (600 and 300 µg/mL, respectively), compared to the concurrent vehicle controls (measured data not reported).
In the Mutation Experiment twelve concentrations, ranging from 5 to 100 µg/mL, were tested in the absence and presence of S-9. No precipitate was observed. Seven days after treatment, the highest two concentrations tested in the absence of S-9 (80 and 100 µg/mL) and the highest concentration tested in the presence of S-9 (100 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition the two lowest concentrations tested in the absence and presence of S-9 (5 and 10 µg/mL) were not selected as there were sufficient concentrations to define the toxicity profile. All other concentrations in the absence and presence of S-9 were selected. The highest concentrations analysed were 70 µg/mL in the absence of S-9 and 80 µg/mL in the presence of S-9, which gave 16 % and 17 % RS, respectively (see Table 2):
Table 2 Mutation Experiment - 3 Hour treatment in the Absence and Presence of S-9
3 Hour Treatment -S-9 |
3 Hour Treatment +S-9 |
||||
Concentration |
% RS |
MF § |
Concentration |
% RS |
MF § |
µg/mL |
|
|
µg/mL |
|
|
0 |
100 |
3.80 |
0 |
100 |
3.05 |
20 |
92 |
7.05NS |
20 |
92 |
5.27 NS |
30 |
102 |
5.88NS |
30 |
95 |
6.60 NS |
40 |
76 |
3.38NS |
40 |
90 |
9.35* |
50 |
64 |
2.42NS |
50 |
76 |
7.32 NS |
55 |
58 |
7.49NS |
55 |
71 |
4.69 NS |
60 |
28 |
4.84NS |
60 |
52 |
6.04 NS |
65 |
24 |
2.77NS |
65 |
46 |
1.80 NS |
70 |
16 |
4.23NS |
70 |
36 |
2.13 NS |
NQO 0.15 |
20 |
65.95 |
80 |
17 |
4.25 NS |
NQO 0.20 |
22 |
89.02 |
B[a]P 2 |
76 |
9.23 |
|
|
|
B[a]P 3 |
69 |
13.86 |
Linear trend tests on mutant frequency -/+ S-9: Not significant (negative trends)
§ 6-TG resistant mutants/106viable cells 7 days after treatment
% RS Percent relative survival adjusted by post treatment cell counts
* Comparison of each treatment with control: Dunnett's test (one-sided), significant at 5% level.
Mutation
A summary of the results for the Mutation Experiment is shown in Table 2. The historical control ranges, based on the last 20 experiments performed in this laboratory, are presented in the Table 3 through Table 4.The acceptance criteria were met and the study was accepted as valid.
Table 4 Vehicle Control Historic Ranges
S-9 |
Mean MF |
MF Range |
|
(Mutants per 106Viable Cells) |
(Mutants per 106Viable Cells) |
-S-9 |
4.67 |
1.15 to 8.20 |
+S-9 |
4.92 |
2.42 to 7.42 |
Table 5 Positive Control Historic Ranges
Control |
S-9 |
Mean MF |
MF Range |
Concentration |
|
(Mutants per 106Viable Cells) |
(Mutants per 106Viable Cells) |
NQO 0.15µg/mL |
-S-9 |
45.51 |
4.04 to 86.97 |
NQO 0.20µg/mL |
-S-9 |
49.20 |
7.90 to 90.49 |
B[a] P 2µg/mL |
+S-9 |
38.37 |
4.06 to 72.69 |
B[a]P 3µg/mL |
+S-9 |
48.54 |
14.95 to 82.13 |
When tested up to toxic concentrations in the absence of S-9, no statistically significant increases in MF, compared to the vehicle control MF value, were observed at any concentration analysed and there was a negative (and non-significant) linear trend.
When tested up to toxic concentrations in the presence of S-9, a statistically significant increase in MF, compared to the vehicle control MF value, was observed at one intermediate concentration (40 µg/mL). The MF value at 40 µg/mL was 9.35 mutants per 106viable cells, compared to the vehicle control MF of 3.05. Although both individual MF values at 40 µg/mL exceeded the historical mean vehicle control MF ± 2 standard deviations (mean 4.92, range 2.42 to 7.42: see Table 4), there were no such increases at any other concentration analysed (up to a maximum of 80 µg/mL, giving 17 % RS) and no evidence of a concentration-related response (the linear trend test was negative over the concentration range tested), therefore this isolated increase in MF was considered of little or no biological relevance.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
No information available.
Endpoint conclusion
- Endpoint conclusion:
- no study available
Mode of Action Analysis / Human Relevance Framework
Not applicable as there were no adverse effects observed.
Additional information
OECD 471 (2017) - In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537 and TA102 of S. typhimuriumwere exposed to Amines, C12-14-branched alkyl dodecylbenzenesulfonates (1:1) in DMSO at concentrations of 5, 16, 50, 160, 500, 1600 and 5000 µg/plate a screening experiment followed by concentrations of 50, 100, 200, 400, 800 and 1600 µg/plate in a secondary, pre-incubation, experiment. Both tests were conducted in the presence and absence of S9 mix. The test item was tested up to cytotoxic and precipitating concentrations. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in the test item treated colonies. This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data
OECD 473 (2017) - In a mammalian cell cytogenetics assay (in vitro chromosome aberration, OECD 473), primary lymphocyte cultures were exposed to Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1) at concentrations of 0, 10, 20, 40, 60, 80, 90, 100, 110, 120, 130, 140, 150 and 175 µg/mL for 3 h with and without metabolic activation. Additionally, a continuous exposure of 24 h was tested without metabolic activation at test item concentrations of 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 80, 100 µg/mL. Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1) was tested up to precipitating and cytotoxic concentrations of 155.5 and 55.99 µg/mL for the 3 h and 20 h exposures, respectively. Positive controls induced the appropriate response. There was no evidence (or a concentration related positive response) of chromosome aberration induction over background values at the highest tested concentration. This study is classified as acceptable. This study satisfies the requirement for Test Guideline In vitro Mammalian Chromosome Aberration Test (OECD 473) in human lymphocyte cells.
OECD 476 (2018) - In a mammalian cell gene mutation assay (HPRT locus of L5178Y), mouse lymphoma cells cultured in vitro were exposed to Amines, C12-14-branched alkyl dodecylbenzenesulfonates (1:1) at concentrations of 20, 30, 40, 50, 55, 60, 65 and 70 µg/mL and 20, 30, 40, 50, 55, 60, 65, 70 and 80 µg/mL in the absence and presence of mammalian metabolic activation (S9-mix), respectively. The test item was tested up to cytotoxic and solubility limits. The positive controls did induce the appropriate response. There was no evidence of induced mutant colonies over background levels at the concentrations tested. This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.
Justification for classification or non-classification
The substance does not meet the criteria for classification in accordance with Regulation (EC) No 1272/2008 (CLP)
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