Registration Dossier
Registration Dossier
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EC number: 226-195-4 | CAS number: 5324-84-5
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Bacterial reverse gene mutation assay: Negative (non-mutagenic); OECD 471; G. Schreib (2018).
In vitro chromosome aberration test: Read-across; source substance - negative (non-mutagenic); OECD 473; C. Flügge (2013).
In vitro mammalian cell gene mutation assay; Read-across, source substance - negative (non-mutagenic); similar to OECD 476; literature publication, McGregor et al. (1988)
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:
- 11 April 2017 - 14 March 2018
- 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 criteria were met.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 21 July 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 30 May 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- August 1998
- Deviations:
- no
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Version / remarks:
- revised 31 March 2011
- 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: Room temperature
- Stability under test conditions: Not specified
- Solubility and stability of the test substance in the solvent/vehicle: Soluble
- 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: The test item was prepared in purified water, diluted prior to treatment and was used within 2 hours of preparation.
- Preliminary purification step (if any): The test item was prepared in purified water.
- Final dilution of a dissolved solid, stock liquid or gel: 50 mg/L
- Final preparation of a solid: N/A
FORM AS APPLIED IN THE TEST (if different from that of starting material) N/A
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:
- The following concentrations (referring to the main constituent) of the test item were prepared and used in the experiments:
Pre-Experiment (Part of Experiment I; Plate-incorporation Test): 2.53, 8.00, 25.3, 80.0, 253, 800, 2000 and 4000 µg/plate (TA 98 and TA 100)
Experiment I (Plate-incorporation Test): 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (TA 1535, TA 1537 and TA 102)
Experiment II (Pre-incubation Test): 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (TA 98, TA 100, TA 1535, TA 1537 and TA 102)
These concentrations correspond to the following concentrations of the test item: Pre-Experiment (Plate-incorporation Test): 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (TA 98 and TA 100)
Experiment I (Plate-incorporation Test): 12.5, 39.5, 125, 395, 1250, 3125 and 6250 µg/plate (TA 1535, TA 1537 and TA 102)
Experiment II (Pre-incubation Test): 12.5, 39.5, 125, 395, 1250, 3125 and 6250 µg/plate (TA 98, TA 100, TA 1535, TA 1537 and TA 102).
5000 µg/plate (concentration of main constituent) was selected as the top dose in the main experiment following the results of the pre-experiment, where it was found that toxic effects occurred at 5000 µg/plate (with and without metabolic activation). It is also the guideline-recommended maximum dose. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: A. dest. (negative controls) or DMSO (positive controls)
- Justification for choice of solvent/vehicle: The chosen solvent A. dest. was compatible with the survival of the bacteria and the S9 activity. DMSO is a guideline recommendation. - Untreated negative controls:
- yes
- Remarks:
- A. dest. (Eurofins Munich, Lot No. 170228 & 170515
- Negative solvent / vehicle controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 4-NOPD: 4-nitro-o-phenylene-diamine; 2-AA: 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium; in agar - plate incorporation (Pre-experiment and experiment I) & preincubation (experiment II)
DURATION
- Preincubation period: 60 min
- Exposure duration (plate incorporation and pre-incubation method): At least 48 h
- Expression time (cells in growth medium): 12 h
- 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: Three plates at each of the eight concentrations tested.
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The Vogel-Bonner Medium E agar plates with 2% glucose used in the Ames Test were prepared by Eurofins Munich.
Vogel-Bonner-salts contain per litre:
10 g MgSO4 x 7 H2O
100 g citric acid
175 g NaNH4HPO4 x 4 H2O
500 g K2HPO4
Sterilisation was performed for 20 min at 121 °C in an autoclave.
Vogel-Bonner Medium E agar plates contain per litre:
15 g Agar Agar
20 mL Vogel-Bonner salts
50 mL glucose-solution (40%)
Sterilisation was performed for 20 min at 121 °C in an autoclave.
The overlay agar contains per litre:
7.0 g Agar Agar
6.0 g NaCl
10.5 mg L-histidine x HCl x H2O
12.2 mg biotin
Sterilisation was performed for 20 min at 121 °C in an autoclave.
Samples of each tester strain were grown by culturing for 12 h at 37 °C in Nutrient Broth to the late exponential or early stationary phase of growth (approx. 10^9 cells/mL). The nutrient medium consists per litre:
8 g Nutrient Broth
5 g NaCl
A solution of 125 µL ampicillin (10 mg/mL) (TA 98, TA 100, TA 102) was added in order to retain the phenotypic characteristics of the strain.
NUMBER OF CELLS EVALUATED: N/A
DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.
- Any supplementary information relevant to cytotoxicity: N/A
OTHER EXAMINATIONS:
N/A - Rationale for test conditions:
- The OECD Guideline for Testing of Chemicals, Section 4, No. 471 - Bacterial Reverse Mutation Test - recommends using a combination of S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.
- Evaluation criteria:
- Criteria of Validity:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100, TA 102)
- the negative control plates (A. dest.) with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range (2014 -2016)):
- S9 + S9
min max min max
TA 98 11 58 15 59
TA 100 49 155 62 160
TA 1535 4 41 3 38
TA 1537 3 35 3 36
TA 102 141 472 157 586
- corresponding background growth on negative control, solvent control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.
Evaluation of Mutagenicity:
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system. - Statistics:
- According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results - a statistical evaluation of the results is not regarded as necessary.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not examined
- Effects of osmolality: Not examined
- Evaporation from medium: Not examined
- Water solubility: Not examined
- Precipitation: No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
- Definition of acceptable cells for analysis: N/A
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100, TA 102)
- the negative control plates (A. dest.) with and without S9 mix are within the historical control data range outliend by Eurofins
- corresponding background growth on negative control, solvent control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.
- Other confounding effects: N/A
RANGE-FINDING/SCREENING STUDIES: N/A
NUMBER OF CELLS WITH MICRONUCLEI : N/A
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Historical Laboratory Control Data of the Positive Control (in 2014 - 2016) without S9 (-S9):
TA 98 TA 100 TA 1535 TA 1537 TA 102
Substance 4-NOPD NaN3 NaN3 4-NOPD MMS
Conc./plate 10 µg 10 µg 10 µg 40 µg 1 µL ≙ 1.3 mg
Mean 430.7 612.1 792.0 94.5 1729.2
SD 155.5 220.0 299.5 22.7 518.8
Min 141 132 38 35 272
Max 1830 1423 1854 273 3321
RSD [%] 36.1 35.9 37.8 24.0 30.0
n 971 1188 931 929 682
Historical Laboratory Control Data of the Positive Control (in 2014 - 2016) with S9 (+S9)
TA 98 TA 100 TA 1535 TA 1537 TA 102
Substance 2-AA 2-AA 2-AA 2-AA MMS
Mean 1880.5 1727.7 133.9 234.1 801.2
SD 708.5 522.0 134.9 101.4 223.7
Min 70 169 22 26 137
Max 3606 3132 1954 682 3588
RSD [%] 37.7 30.2 100.8 43.3 27.9
n 966 1184 927 925 678
- Negative (solvent/vehicle) historical control data:
Historical Laboratory Control Data of the Negative Control (in 2014 - 2016) without S9 (-S9):
TA 98 TA 100 TA 1535 TA 1537 TA 102
Mean 24.2 90.7 13.8 8.2 270.4
SD 6.7 15.6 6.7 2.9 55.0
Min 11 49 4 3 141
Max 58 155 41 35 472
RSD [%] 27.7 17.2 48.6 35.3 20.3
n 972 1191 929 931 682
Historical Laboratory Control Data of the Negative Control (in 2014 - 2016) with S9 (+S9):
TA 98 TA 100 TA 1535 TA 1537 TA 102
Mean 29.0 96.4 10.5 8.3 339.7
SD 6.8 14.1 4.5 3.1 71.3
Min 15 62 3 3 157
Max 59 160 38 36 586
RSD [%] 23.4 14.6 42.7 37.4 21.0
n 967 1189 925 926 676
ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxic effects of the test item were noted in tester strain TA 102 up to the highest dose group evaluated (with and without metabolic activation) in experiment I and II.
Toxic effects of the test item were noted in all other tester strains evaluated in the pre-experiment, experiment I and II.
In the pre-experiment toxic effects of the test item were observed in tester strain TA 98 at concentrations of 2000 µg/plate and higher (without metabolic activation) and at a concentration of 4000 µg/plate (with metabolic activation). In tester strain TA 100 toxic effects of the test item were noted at concentrations of 800 µg/plate and higher (without metabolic activation) and at a concentration of 4000 µg/plate (with metabolic activation).
In experiment I toxic effects of the test item were seen in tester strain TA 1535 at a concentration of 5000 µg/plate (without metabolic activation). In tester strain TA 1537 toxic effects of the test item were observed at a concentration of 5000 µg/plate (with and without metabolic activation).
In experiment II toxic effects of the test item were noted in tester strain TA 98 at a concentration of 5000 µg/plate (with and without metabolic activation). In tester strain TA 100 toxic effects of the test item were seen at concentrations of 2500 µg/plate and higher (without metabolic activation) and at a concentration of 5000 µg/plate (with metabolic activation). In tester strains TA 1535 and TA 1537 toxic effects of the test item were observed at concentrations of 2500 µg/plate and higher (with and without metabolic activation). - Conclusions:
- The test item is considered to be non-mutagenic under the experimental conditions of this bacterial reverse mutation assay.
The substance does not meet the criteria for classfication in accordance with GHS or Regulation (EC) No 1272/2008 (CLP). - Executive summary:
In accordance with OECD 471, the test item sodium octane-1-sulphonate was tested for its potential to induce gene mutations. A plate incorporation test and pre-incubation test was conducted with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.
In three independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate.
The following concentrations referring to the main constituent of the test item were prepared and used in the experiments:
Pre-Experiment (Part of Experiment I):
2.53, 8.00, 25.3, 80.0, 253, 800, 2000 and 4000 µg/plate (TA 98 and TA 100)
Experiment I:
10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (TA 1535, TA 1537 and TA 102)
Experiment II:
10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (TA 98, TA 100, TA 1535, TA 1537 and TA 102)
These concentrations correspond to the following concentrations of the test item:
Pre-Experiment (Part of Experiment I):
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (TA 98 and TA 100)
Experiment I:
12.5, 39.5, 125, 395, 1250, 3125 and 6250 µg/plate (TA 1535, TA 1537 and TA 102)
Experiment II:
12.5, 39.5, 125, 395, 1250, 3125 and 6250 µg/plate (TA 98, TA 100, TA 1535, TA 1537 and TA 102).
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
No toxic effects of the test item were noted in tester strain TA 102 up to the highest dose group evaluated (with and without metabolic activation) in experiment I and II.
Toxic effects of the test item were noted in all other tester strains evaluated in the pre-experiment, experiment I and II.
In the pre-experiment toxic effects of the test item were observed in tester strain TA 98 at concentrations of 2000 μg/plate and higher (without metabolic activation) and at a concentration of 4000 μg/plate (with metabolic activation). In tester strain TA 100 toxic effects of the test item were noted at concentrations of 800 μg/plate and higher (without metabolic activation) and at a concentration of 4000 μg/plate (with metabolic activation).
In experiment I toxic effects of the test item were seen in tester strain TA 1535 at a concentration of 5000 μg/plate (without metabolic activation). In tester strain TA 1537 toxic effects of the test item were observed at a concentration of 5000 μg/plate (with and without metabolic activation).
In experiment II toxic effects of the test item were noted in tester strain TA 98 at a concentration of 5000 μg/plate (with and without metabolic activation). In tester strain TA 100 toxic effects of the test item were seen at concentrations of 2500 μg/plate and higher (without metabolic activation) and at a concentration of 5000 μg/plate (with metabolic activation). In tester strains TA 1535 and TA 1537 toxic effects of the test item were observed at concentrations of 2500 μg/plate and higher (with and without metabolic activation).
No biologically relevant increases in revertant colony numbers in any of the five tester strains were observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. All criteria of validity were met. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 4 October 2012 - 27 March 2013
- 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 criteria were met.
- Justification for type of information:
- Read-across justification document is provided in Section 13.2.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- 21 July 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- 30 May 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: CH Guideline S2 (R1): 'Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use (EMA/CHMP/ICH/126642/2008)'
- Version / remarks:
- Accounting for planned revisions to the guidelines
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: in vitro mammalian chromosome aberration test (migrated information)
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Confidential
- Expiration date of the lot/batch: 12 December 2013
- Purity test date: Not specified
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: At +15°C to +25°C, below 70% relative humidity.
- Stability under test conditions: Not specified
- Solubility and stability of the test substance in the solvent/vehicle: soluble in aqua ad iniectabilia at 5000 μg/ml
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A (fresh preparations of the test item were used for all experimental parts)
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: the test item was completely dissolved in aqua ad iniectabilia (with a correction factor of 1.02 applied due to a purity of 98%) and appropriate serial dilutions prepared.
- Final preparation of a solid: N/A
FORM AS APPLIED IN THE TEST (if different from that of starting material) As liquid
OTHER SPECIFICS: N/A - Species / strain / cell type:
- lymphocytes: human peripheral
- Remarks:
- Human peripheral blood was obtained by venepuncture from healthy donors known to be without any medication and collected in heparinised vessels
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Healthy human donor.
- Whether whole blood or separated lymphocytes were used if applicable: Small innocula of whole blood (0.5 mL) were added to tubes containing 5 mL of complete culture medium.
- Sex, age and number of blood donors if applicable: One donor per exposure group, i.e. one donor for the duplicate cultures of the -S9 group and a different donor for the duplicate cultures of the +S9 group.
- Methods for maintenance in cell culture if applicable: The tubes were sealed and incubated at 37°C with occasional shaking to prevent clumping.
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- (With and without metabolic activation, 4h or 24 h exposure)
125, 250, 500, 1000, 1500, 2000 or 2500 μg test item/mL medium
Justification:
In the preliminary experiment, cytotoxicity was noted at 2500 and 5000 μg test item/mL with and without metabolic activation (4-h or 24-h exposure). Hence, the high test concentration employed in the main study was 2500 μg test item/mL medium in the experiments without and with metabolic activation. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: aqua ad iniectabilia
- Justification for choice of solvent/vehicle: OECD Guideline recommended - Untreated negative controls:
- yes
- Remarks:
- aqua ad iniectabilia
- Negative solvent / vehicle controls:
- yes
- Remarks:
- aqua ad iniectabilia
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in suspension
- Cell density at seeding (if applicable): 0.5ml whole blood
DURATION
- Preincubation period: 48 h
- Exposure duration: Two exposure times without S9 mix: 4 and 24 h , and 1 exposure time with S9 mix: 4 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): Harvesting time was 24 h after exposure
SELECTION AGENT (mutation assays): N/A
SPINDLE INHIBITOR (cytogenetic assays): Two hours before termination the cell division was arrested by the addition of 0.5 mL of a 10 μg/mL solution of the spindle poison colcemid to each culture.
STAIN (for cytogenetic assays): Giemsa stain
NUMBER OF REPLICATIONS: The study was conducted in duplicate.
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The tubes were centrifuged, the fixative removed and the cell pellet resuspended in a few drops of 60% acetic acid. Single drops of the cell suspension were spread on clean, grease-free glass slides on a hot plate (approx. 50°C) and the slides were left to air-dry. Two slides were prepared per culture, stained for 30 minutes in Giemsa stain (1:10 in WEISE's buffer 7 pH 6.8), washed in buffer and left to air-dry. Permanent slides were made using CONSUL MOUNT mountant after clearing in xylene.
NUMBER OF CELLS EVALUATED: To determine the test item toxicity, 1000 cells were scored and the mitotic index was calculated as the percentage of cells in metaphase.
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): For each treatment and culture 100 metaphases were examined, if possible.
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: N/A
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
- Any supplementary information relevant to cytotoxicity:
A preliminary cytotoxicity study was conducted to establish the top concentration for the main cytogenetic test. Concentrations of 10, 25, 100, 250, 1000, 2500 and 5000 μg Bio-Terge PAS 7S-DRY/mL medium were employed in an experiment without and with metabolic activation. In this preliminary experiment cytotoxicity was noted at 2500 and 5000 μg Bio-Terge PAS 7S-DRY/mL in the experiment with and without metabolic activation (4-h or 24-h exposure). In the main study cytotoxicity was noted in the experiments without and with metabolic activation at the top concentration of 2500 μg Bio-Terge PAS 7S-DRY/mL medium.
OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): N/A
- OTHER:
-pH and osmolaity:
The pH and osmolality of the negative control and all test item formulations in the medium were determined for each experiment employing the methods given below:
pH values: using a digital pH meter type WTW pH 525 (series no. 51039051), Osmolality: with a semi-micro osmometer.
-Metaphase evaluation:
The total number of gaps was recorded in 100 metaphases for each culture, if possible. Metaphases which differed from the normal diploid complement (46) were excluded from evaluation. However, test item-related variations of the normal chromosome number were noted (polyploidy / endoreduplication).
Metaphases were assigned to one of the following 5 categories:
1 = normal metaphases
2 = metaphases with 1 - 2 aberrations
3 = metaphases with multiple aberrations
4 = pulverised metaphases: extreme (=>50%) fragmentation of chromosomal material
5 = polyploidy / endoreduplication
Observed aberrations were noted and scored according to J. R. K. SAVAGE (1975):
Gap: Achromatic region in chromatid(s) not greater than the width of a chromatid; scored as single-stranded or double-stranded.
Break: Achromatic region in chromatid(s) greater than the width of a chromatid or a discontinuity with displacement; scored as chromatid or chromosomal.
Fragment: Any free displaced portion of chromatid material.
Exchange: Aberration arising from an exchange between two or more chromosomes which results in the products re-uniting to form a dicentric or polycentric structure. These may be chromosome or chromatid interchanges. In studies of this type, where full karyotyping is not undertaken and chromosome banding has not been performed, only asymmetrical or chromatid exchanges will normally be recognised.
Other types of aberrations: E.g. isochromatid/isochromatid exchanges such as dicentric chromosomes, centric rings, pulverised metaphases or polyploidy. - Evaluation criteria:
- The test item is judged to have mutagenic properties with respect to chromosomal or chromatid change, if the following criteria are fulfilled:
- the number of chromosomal aberrations is significantly (at p => 0.05) increased compared with the solvent control
- the increase observed is concentration-dependent
- both duplicate cultures provide similar results
- the increase should not occur in the severely cytotoxic range (mitotic index <0.25), as it is known that high cytotoxicity may cause artefacts in the form of aberrations in in vitro chromosomal aberration tests
- a reproducible increase in the number of cells with chromosomal aberrations. - Statistics:
- The assessment was carried out by a comparison of the number of chromosome aberrations of the samples with those of the solvent control, using the exact test of R. A. FISHER (p => 0.05) as recommended by the UKEMS guidelines (The United Kingdom Branch of the European Environmental Mutagen Society: Report of the UKEMS subcommittee on guidelines for mutagenicity testing, part III, Statistical evaluation of mutagenicity test data, 1989).
It is generally accepted that chromatid gaps are not examples of true chromosomal aberrations. In this study, therefore only the total numbers of cells with aberrations and without gap damage were analysed. However, the numbers of cells with aberra- tions including gap damage are also tabulated. - Key result
- Species / strain:
- lymphocytes: human peripheral
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative 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 specified
- Water solubility: None
- Precipitation: None
- Definition of acceptable cells for analysis: Metaphases which differed from the normal diploid complement were excluded from evaluation.
- Other confounding effects: N/A
RANGE-FINDING/SCREENING STUDIES:
A preliminary cytotoxicity study was conducted to establish the top concentration for the main cytogenetic test. Concentrations of 10, 25, 100, 250, 1000, 2500 and 5000 μg test item/mL medium were employed in an experiment without and with metabolic activation. In this preliminary experiment cytotoxicity was noted at 2500 and 5000 μg test item/mL in the experiment with and without metabolic activation (4-h or 24-h exposure). Hence, the highest concentration employed in the main study was 2500 μg Test item/mL medium in the experiments without and with metabolic activation.
CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: N/A
NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: N/A
- Indication whether binucleate or mononucleate where appropriate: N/A
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Yes
- Negative (solvent/vehicle) historical control data: Yes
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Miotic index
- Other observations when applicable: N/A
Determination of polyploidy: No test item-related polyploidy was noted in the experiments without or with metabolic activation.
Determination of endoreplication: No test item-related endoreduplication were noted in the experiments without or with metabolic activation. - Conclusions:
- Under the conditions of this study, the test item was considered not to present mutagenic properties with respect to chromosomal or chromatid damage.
- Executive summary:
A test according to OECD Guideline 473 was conducted in order to determine whether the test item caused structural chromosomal aberrations in cultured mammalian cells.
Samples of the test item were assayed in an in vitro cytogenetic study using human lymphocyte cultures both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced animals.
The test was carried out employing two exposure times without S9 mix: 4 and 24 hours, and one exposure time with S9 mix: 4 hours. The experiment with S9 mix was carried out twice. The harvesting time was 24 hours after starting of exposure. The incubation procedure took place in the dark. The study was conducted in duplicate.
The test item was completely dissolved in aqua ad iniectabilia. A correction factor of 1.02 was used as the purity of test item was 98% only. The vehicle served as the negative control.
Tests without metabolic activation (4- and 24-hour exposure)
The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with test item at concentrations of 125, 250, 500, 1000, 1500, 2000 or 2500 μg test item/mL medium (4-h or 24-h exposure) in the absence of metabolic activation ranged from 1.0% to 3.5%. These results were within the range of the historical control data (0 - 4%).
The result for the vehicle control cultures was a mean of 1% cells with aberrations (excluding gaps), which is within the historical control range. The positive control cultures had a significantly increased frequency of cells with aberrations, in line with the historical control range. Therefore, the test is considered to be valid.
Test with metabolic activation (4-hour exposure)
The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with test item at concentrations of 125, 250, 500, 1000, 1500, 2000 or 2500 μg test item /mL medium in the presence of metabolic activation in the first and second experiment ranged from 0.5% to 4.0%. These results were within the range of the historical control data (0 - 4%).
The result for the vehicle control cultures was a mean of 1% cells with aberrations (excluding gaps), which is within the historical control range. The positive control cultures had a significantly increased frequency of cells with aberrations, in line with the historical control range. Therefore, the test is considered to be valid. No test item-related polyploidy or endoreduplication were noted in the experiments without or with metabolic activation.
Under the conditions of the study, tested up to a cytotoxic concentration of 2500 μg test item/mL medium, in the absence and in the presence of metabolic activation, employing two exposure times (without S9) and one exposure time (with S9), revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage.
In the same test, Mitomycin C and cyclophosphamide induced significant increases in the frequency of damaged cells, which confirmed the validity of this assay.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
Read-across justification document is provided in Section 13.2. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- lymphocytes: human peripheral
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Under the conditions of this study, the test item was considered not to present mutagenic properties with respect to chromosomal or chromatid damage. This result is appropriate under a read-across approach for applicability to the target substance sodium octane-1-sulphonate monohydrate.
- Executive summary:
An OECD Test Guideline 473 was conducted in order to determine whether the test item caused structural chromosomal aberrations in cultured mammalian cells.
Samples of the test item were assayed in anin vitrocytogenetic study using human lymphocyte cultures both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced animals.
The test was carried out employing two exposure times without S9 mix: 4 and 24 hours, and one exposure time with S9 mix: 4 hours. The experiment with S9 mix was carried out twice. The harvesting time was 24 hours after starting of exposure. The incubation procedure took place in the dark. The study was conducted in duplicate.
The test item was completely dissolved in aqua ad iniectabilia. A correction factor of 1.02 was used as the purity of test item was 98% only. The vehicle served as the negative control.
Tests without metabolic activation (4- and 24-hour exposure)
The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with test item at concentrations of 125, 250, 500, 1000, 1500, 2000 or 2500 μg test item/mL medium (4-h or 24-h exposure) in the absence of metabolic activation ranged from 1.0% to 3.5%. These results were within the range of the historical control data (0 - 4%).
The result for the vehicle control cultures was a mean of 1% cells with aberrations (excluding gaps), which is within the historical control range. The positive control cultures had a significantly increased frequency of cells with aberrations, in line with the historical control range. Therefore, the test is considered to be valid.
Test with metabolic activation (4-hour exposure)
The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with test item at concentrations of 125, 250, 500, 1000, 1500, 2000 or 2500 μg test item /mL medium in the presence of metabolic activation in the first and second experiment ranged from 0.5% to 4.0%. These results were within the range of the historical control data (0 - 4%).
The result for the vehicle control cultures was a mean of 1% cells with aberrations (excluding gaps), which is within the historical control range. The positive control cultures had a significantly increased frequency of cells with aberrations, in line with the historical control range. Therefore, the test is considered to be valid. No test item-related polyploidy or endoreduplication were noted in the experiments without or with metabolic activation.
Under the conditions of the study, tested up to a cytotoxic concentration of 2500 μg test item/mL medium, in the absence and in the presence of metabolic activation, employing two exposure times (without S9) and one exposure time (with S9), revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage.
In the same test, Mitomycin C and cyclophosphamide induced significant increases in the frequency of damaged cells, which confirmed the validity of this assay.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1987 (revised in 1988)
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to Section 13.2 for read-across justification document. - Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- not applicable
- GLP compliance:
- not specified
- Type of assay:
- other: in vitro mammalian cell gene mutation tests using the thymidine kinase gene (migrated information)
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: National Toxicology Program Chemical Repository, Radian Corporation, Austin, TX 78766
- Expiration date of the lot/batch: Not specified
- Purity test date: Not specified
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: Not specified
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: Soluble
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None reported
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: 0.1 mg/L
- Final preparation of a solid: N/A
FORM AS APPLIED IN THE TEST (if different from that of starting material) N/A
TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) N/A
OTHER SPECIFICS: - Target gene:
- N/A
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- The tk+/tk- -3.7.2C heterozygote of L5178Y mouse lymphoma cells (in the presence of 5-trifluorothymidine (TFT) was obtained from Dr.D. Clive, Burroughs Wellcome Co., Research Triangle Park, NC 27709, and stored in liquid nitrogen
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9
- Test concentrations with justification for top dose:
- (-S9)
Trial 1: 0, 3.125, 6.25, 12.5, 25, 50, 100 µg/ml
Trial 2: 0, 10, 20, 30, 40, 50, 60, 70 µg/mL
Trial 3: 0, 50, 55, 60, 65, 70 µg/mL
Trial 4: 0, 30, 40, 50, 60, 70, 80 µg/mL
Trial 5: 0, 50, 55, 60, 65, 70 µg/mL
(+S9)
Trial 6: 0, 50, 60, 70, 80, 90 µg/mL
Trial 7: 0, 55, 65, 75, 85, 95 µg/mL
Trial 8: 0, 60, 65, 70, 75, 80 µg/mL
(No justification provided for top dose) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Analytical grade DMSO
- Justification for choice of solvent/vehicle: Not specified (however this solvent is compliant with OECD Test Guideline 476) - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO (suppplied by BDH Limited, Poole, Dorset, England)
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- Supplied by Radian Corporation, Austin, TX 78766
- Positive control substance:
- 3-methylcholanthrene
- methylmethanesulfonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 6 x 10^6 in a final volume of 10ml F5p in a 30-ml screw-cap plastic tube (Sterilin Ltd.)
DURATION
- Preincubation period: 4 h
- Exposure duration: 48 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): The agar was gelled at 4°C for 5-10 min, then the plates were incubated for 11-14 days in 5% CO2:95% air at 37°C
- Fixation time (start of exposure up to fixation or harvest of cells):
SELECTION AGENT (mutation assays): 3 µg trifluorothymidine/mL
SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for cytogenetic assays): Not specified
NUMBER OF REPLICATIONS: Two
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Thawed samples were cultured following storage in liquid nitrogen. Laboratory cultures were confimed as free from mycoplasma by cultivating or Hoechst staining techniques and maintained in Fischer's medium at 37 °C on gyratory tables. Fischer's medium (designated F0) was supplemented with 2 mM L-glutamine, sodium pyruvate, 110 µg/mL, 0.05% pluronic F68, antibiotics, and 10% heat-inactivated donor horse serum (v/v) (designated F10p).On a single occasion, within 1 week of the start of an experiment, cultures were purged of tk-/tk- mutants by exposure for 1 day to F10p containing THMG (thymidine, 6 µg/mL hypoxanthine, 5 µg/mL, glycine, 7.5 µg/mL and methotrexate, 0.1 µg/mL), then for 3 days to F10p containing THG only, (i.e., THMG without methotrexate).
NUMBER OF CELLS EVALUATED:
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: cloning efficiency; relative total growth: A 01-mL sample of the cell suspension was withdrawn and diluted 1:100. Three 0.1-mL samples (200 cells) of the diluted cultures were transferred to 30-mL tubes, mixed with 25-ml cloning medium (Fischer's medium containing 20% heat-inactivated horse serum, i.e., F20p) containing 0.35% Noble agar and poured into 90-mm Petri plates).
Toxicity was expressed as either a reduction of cell population growth in suspension during the expression period or a reduction in cloning efficiency. A measure of the overall toxicity was the relative total growth (RTG), which is defined as:
RTG = (total suspension growth x cloning efficiency) in dosed culture / (total suspension growth x cloning efficiency) in control culture
Mutant fraction (MF) was calculated as follows:
MF = 200 x mutant clones per plate (usually a mean of 3) / total clones per plate (usually a mean of 3)
= mutants/10^6 clonable cells.
- Any supplementary information relevant to cytotoxicity: The first experiment was a toxicity test in which cell population expansion was measured. Ten-fold differences in test compound concentrationswere used in the toxicity test, the highest being 0.1 mg/ml dictated by the solubility of the test item
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:
Colony counting: Colonies were counted using an Artek 880 Automated Colony Counter, with the colony size discriminator control in the “off” position.
Exposure: Each exposed culture consisted of 6 x 10^6 cells in a volume of 10ml F5p in a 30-ml screw-cap plastic tube (Sterilin Ltd.).This tube was incubated for 4 hr on a horizontal axis roller drum rotating at 10 rpm. At the end of the incubation time, the cells were sedimented by centrifugation at 500 g.av. for 10 min, washed, and finally resuspended in 20 mL F10p. These cell suspensions (3 x 10^5 cell/mL) were incubated for a 2-day expression period, the cell population density being adjusted back to 20 mL of 3 x 10^5 cells/mL after 24 h. After 48 h, the cell population densities were estimated and culture volumes containing 3 x 10^6 cells adjusted to 15 mL with F10p, giving a cell population density of 2 x 10^5 cells/mL.
Mutant selection: Three aliquots (each containing 10^6 cells) of the remaining culture were distributed to 30-mL tubes, mixed with 20-mL cloning medium to give final concentrations of 0.35% Noble agar and 3 µg trifluorothymidine/mL, then poured into 90-mm Petri plates. - Evaluation criteria:
- 1. SOLVENT CONTROL
a) A solvent control count was rejected if the cloning efficiency was <50% or >115%. Between 50 and 60%, judgment was used in accepting the result.
b) The average mutant fraction of the solvent controls had to be >15 mutants per 10^6 surviving cells and <110 mutants per 10^6 surviving cells. The range was extended to >10 - <150mutants per 10^6 surviving cells for experiments that were positive.
c) Unless at least two solvent control cultures were accepted, the experiment was rejected.
d) The experiment was rejected if a chi-square test for consistency of the acceptable mutant
fractions showed P<5%.
2. POSITIVE CONTROL
a) A positive control culture was rejected if the cloning efficiency was <10% or >115%.
b) A positive control culture was rejected if the relative total growth (RTG) was <1%.
3. DOSES
a) A culture was rejected if the cloning efficiency was <10% or >115%.
b) A culture was rejected if the RTG was <1%.
c) A culture was rejected if the relative suspension growth for the second day of expression was 40%.
d) If the RTG was between 1 and 5% and/or the cloning efficiency was between 10 and 20%, then the mutant count was examined. If a significant increase in the mutant fraction was not supported by an increase in the mutant count, then the culture was rejected.
e) A culture was rejected if the day two count was <3 x 10^5 per ml.
f) A dose was rejected if the compound was not soluble at that dose.
g) No dose greater than 5 mg/ml was tested.
h) A dose set was rejected if the chi-square test for consistency of the acceptable mutant fractions within that dose set showed P<5%.
i) Each dose set had to contain two or more acceptable cultures.
j) If fewer than three dose sets were accepted, then the experiment was rejected unless the reason for dose set rejection was precipitation and there was no mutagenic response. - Statistics:
- The statistical analysis was based upon the mathematical model proposed for this system (Lee and Caspary, 1983) and consisted of a dose-trend test (Barlow et al., 1972) and a variance analysis of pair-wise comparisons of each dose against the vehicle control
Lee YJ, Caspary WJ (1983): Mathematical model of L5178Y mouse lymphoma forward mutation assay. Mutat Res 113:417-430.
Barlow RE, Bartholomew CJ, Bremner JM, Brunk HD (1972):“Statistical Inference Under Order Restrictions: The Theory and Application of Isotonic Regression.” New York: John Wiley and Sons, p 215. - 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 applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None
- Effects of osmolality: Not measured
- Evaporation from medium: Not specified
- Water solubility: Not specified
- Precipitation: None
- Definition of acceptable cells for analysis:
- Other confounding effects: N/A
RANGE-FINDING/SCREENING STUDIES: N/A
CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: N/A
NUMBER OF CELLS WITH MICRONUCLEI: N/A
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Not provided
- Negative (solvent/vehicle) historical control data: Not provided
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; RICC, RPD or PI when cytokinesis block is not used]
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells] N/A - Conclusions:
- Under the conditions of this test, it is concluded that the test item is not mutagenic. This result is appropriate under a read-across approach for applicability to the target substance sodium octane-1-sulphonate monohydrate.
- Executive summary:
A mammalian cell gene mutation assay using mouse lymphoma L5178Y cells, in a test that was similar to an OECD 476 In Vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes (latest version adopted 29 July 2016), was performed using the test substance sodium dodecyl sulphate (read-across source substance for target substance: sodium octane-1-sulphonate monohydrate).
The test item was tested in both the absence and presence of metabolic activation (S9 mix) at concentrations of: -S9: 3.125, 6.25, 10, 12.5, 20, 25, 30, 40, 50, 55, 60, 65, 70, 80 and 100 μg/mL, +S9: 50, 55, 60, 65, 70, 75, 80, 85, 90 and 95 μg/mL.
Eight acceptable experiments were conducted, five in the absence of S9 mix. In the first of these, statistically significant increases in mutant fraction were observed at three dose levels: 6.25, 25, and 50 µg/ml; 100 µg/ml was a lethal concentration. Over the nonlethal range, there were generally elevated mutant fractions, the highest being 1.9-fold the control level at 25 µg/ml. Although these increases in mutant fraction were significant, the lack of an obvious dose-related response with a relatively soluble chemical over a dose range which was not toxic encourages speculation that the increases were not due to the test material.
In the second experiment without S9 mix, there was a clearly significant response at 60 µg/ml, but at no other dose. The RTG was about 22%. Experiment 3 gave a statistically significant response (1.7-fold increase) at60µg/ml,but not at 65 µg/ml. The mutant fraction at 70 µg/ml was only 44/10^6 survivors, so this single culture result supports the view that the statistically significant result at the lower dose level was a chance event. The succeeding experiments without S9 mix were unambiguously negative, as were two experiments with S9 mix. The last experiment with S9 mix was inconclusive because the RTG at 80 µg/mL was about 86% and there was no indication of a mutagenic response.
Although the higher dose levels of the test item induced toxic effects in the cells, there was no enhanced mutation rate in the treated or untreated cells, either with or without metabolic activation. The positive controls induced the expected mutagenic response and therefore the test was considered to be valid. Therefore, the test substance was considered to be non-mutagenic.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
See justification in Section 13.2. - Reason / purpose for cross-reference:
- read-across source
- 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 applicable
- Positive controls validity:
- valid
- Conclusions:
- Under the conditions of this test, it is concluded that the test item is not mutagenic.
- Executive summary:
A mammalian cell gene mutation assay using mouse lymphoma L5178Y cells, in a test that was similar to an OECD 476In VitroMammalian Cell Gene Mutation Tests using theHprtandxprtgenes (latest version adopted 29 July 2016), was performed using the test substance sodium dodecyl sulphate (read-across source substance for target substance: sodium octane-1-sulphonate monohydrate).
The test item was tested in both the absence and presence of metabolic activation (S9 mix) at concentrations of: -S9: 3.125, 6.25, 10, 12.5, 20, 25, 30, 40, 50, 55, 60, 65, 70, 80 and 100 μg/mL, +S9: 50, 55, 60, 65, 70, 75, 80, 85, 90 and 95 μg/mL.
Eight acceptable experiments were conducted, five in the absence of S9 mix. In the first of these, statistically significant increases in mutant fraction were observed at three dose levels: 6.25,25,and 50 µg/ml; 100 µg/ml was a lethal concentration. Over the nonlethal range, there were generally elevated mutant fractions, the highest being 1.9-fold the control level at 25 µg/ml. Although these increases in mutant fraction were significant, the lack of an obvious dose-related response with a relatively soluble chemical over a dose range which was not toxic encourages speculation that the increases were not due to the test material.
In the second experiment without S9 mix, there was a clearly significant response at 60 µg/ml, but at no other dose. The RTG was about 22%. Experiment 3 gave a statistically significant response (1.7-fold increase) at60µg/ml,but not at 65 µg/ml. The mutant fraction at 70 µg/ml was only 44/10^6 survivors, so this single culture result supports the view that the statistically significant result at the lower dose level was a chance event. The succeeding experiments without S9 mix were unambiguously negative, as were two experiments with S9 mix. The last experiment with S9 mix was inconclusive because the RTG at 80 µg/mL was about 86% and there was no indication of a mutagenic response.
Although the higher dose levels of the test item induced toxic effects in the cells, there was no enhanced mutation rate in the treated or untreated cells, either with or without metabolic activation. The positive controls induced the expected mutagenic response and therefore the test was considered to be valid. Therefore, the test substance was considered to be non-mutagenic.
Referenceopen allclose all
Table 1: Pre-experimental Results (Plate-incorporation Test)
Treatment |
Dose (µg/plate) |
Mutation factor (toxicity) |
|
Without S9 |
With S9 |
||
TA 98 |
|||
Test item |
2.53 |
1.1 |
1.1 |
8.00 |
1.0 |
0.8 |
|
25.3 |
1.2 |
1.1 |
|
80 |
1.4 |
0.8 |
|
253 |
1.2 |
0.6 |
|
800 |
1.5 |
0.8 |
|
2000 |
0.4 [B] |
0.7 |
|
4000 |
0.6 [B] |
0.5 |
|
A dest. |
- |
1.0 |
1.0 |
4-NOPD |
10 |
21.2 |
- |
NaN3 |
10 |
- |
- |
2-AA |
2.5 |
- |
54.4 |
TA 100 |
|||
Test item |
2.53 |
1.0 |
0.8 |
8.00 |
1.1 |
1.0 |
|
25.3 |
1.2 |
0.9 |
|
80 |
1.0 |
0.9 |
|
253 |
0.8 |
0.8 |
|
800 |
0.6 [B] |
1.0 |
|
2000 |
0.6 [B] |
1.0 |
|
4000 |
0.4 [B] |
0.8 [B] |
|
A dest. |
- |
1.0 |
1.0 |
4-NOPD |
10 |
- |
- |
NaN3 |
10 |
7.2 |
- |
2-AA |
2.5 |
- |
24.1 |
*(toxicity parameter): B = Background lawn reduced; N = No background lawn
Dose refers to concentration of main constituent.
Table 2: Experiment I Results (Plate-incorporation Test)
Treatment |
Dose (µg/plate) |
Revertant colonies per plate |
Mutation factor |
||||
Without S9 (mean) |
SD |
With S9 (mean) |
SD |
-S9 |
+S9 |
||
TA 98 |
|||||||
Test item |
2.53 |
25 |
2.6 |
28 |
9.1 |
1.1 |
1.1 |
8.00 |
23 |
6.8 |
22 |
6.7 |
1.0 |
0.8 |
|
25.3 |
27 |
4.6 |
27 |
0.6 |
1.2 |
1.1 |
|
80 |
31 |
4.4 |
21 |
7.5 |
1.4 |
0.8 |
|
253 |
26 |
1.2 |
15 |
4.0 |
1.2 |
0.6 |
|
800 |
33 |
2.1 |
22 |
7.2 |
1.5 |
0.8 |
|
2000 |
9 [B] |
8.5 |
18 |
7.9 |
0.4 |
0.7 |
|
4000 |
13 [B] |
2.5 |
13 |
6.7 |
0.6 |
0.5 |
|
A dest. |
- |
22 |
8.5 |
26 |
4.0 |
1.0 |
1.0 |
4-NOPD |
10 |
466 |
77.5 |
/ |
/ |
21.2 |
/ |
2-AA |
2.5 |
/ |
/ |
1415 |
333.4 |
/ |
54.4 |
TA 100 |
|||||||
Test item |
2.53 |
116 |
15.7 |
69 |
5.5 |
1.0 |
0.8 |
8.00 |
133 |
2.5 |
86 |
3.5 |
1.1 |
1.0 |
|
25.3 |
141 |
12.5 |
83 |
17.9 |
1.2 |
0.9 |
|
80 |
126 |
8.6 |
77 |
13.1 |
1.0 |
0.9 |
|
253 |
94 |
5.0 |
73 |
4.6 |
0.8 |
0.8 |
|
800 |
68 [B] |
18.8 |
90 |
17.9 |
0.6 |
1.0 |
|
2000 |
72 [B] |
5.5 |
91 |
12.9 |
0.6 |
1.0 |
|
4000 |
47 [B] |
2.5 |
75 [B] |
3.1 |
0.4 |
0.8 |
|
A dest. |
- |
121 |
4.0 |
89 |
19.6 |
1.0 |
1.0 |
NaN3 |
10 |
874 |
81.1 |
/ |
/ |
7.2 |
/ |
2-AA |
2.5 |
/ |
/ |
2152 |
201.3 |
/ |
24.1 |
TA 1535 |
|||||||
Test item |
10 |
19 |
5.1 |
17 |
1.7 |
1.1 |
1.1 |
31.6 |
16 |
2.9 |
16 |
1.0 |
0.9 |
1.0 |
|
100 |
18 |
6.1 |
15 |
5.9 |
1.1 |
1.0 |
|
316 |
22 |
3.5 |
13 |
3.1 |
1.3 |
0.8 |
|
1000 |
17 |
0.0 |
12 |
3.5 |
1.0 |
0.8 |
|
2500 |
17 |
2.6 |
14 |
4.0 |
1.9 |
0.9 |
|
5000 |
4 [B] |
1.0 |
14 |
4.7 |
0.2 |
0.9 |
|
lA dest. |
- |
17 |
46.5 |
16 |
4.4 |
1.0 |
1.0 |
NaN3 |
10 |
1010 |
46.5 |
/ |
/ |
60.6 |
/ |
2-AA |
2.5 |
/ |
/ |
110 |
13.9 |
/ |
6.9 |
TA 1537 |
|||||||
Test item |
10 |
11 |
1.5 |
9 |
1.2 |
1.0 |
1.0 |
31.6 |
10 |
0.6 |
9 |
0.6 |
0.9 |
1.0 |
|
100 |
12 |
0.6 |
8 |
1.2 |
1.1 |
0.9 |
|
316 |
13 |
1.2 |
8 |
0.6 |
1.1 |
1.0 |
|
1000 |
12 |
1.0 |
8 |
1.2 |
1.0 |
1.0 |
|
2500 |
11 |
1.0 |
6 |
0.6 |
0.9 |
0.7 |
|
5000 |
2 [B] |
1.5 |
1 [B] |
1.0 |
0.2 |
0.1 |
|
A dest. |
- |
12 |
0.6 |
9 |
0.6 |
1.0 |
1.0 |
4-NOPD |
40 |
100 |
15.4 |
/ |
8.6 |
8.6 |
0 |
2-AA |
2.5 |
/ |
/ |
268 |
/ |
/ |
31.0 |
TA 102 |
|||||||
Test item |
10 |
303 |
13.0 |
353 |
43.7 |
0.9 |
0.9 |
31.6 |
319 |
11.9 |
366 |
5.1 |
0.9 |
0.9 |
|
100 |
332 |
16.8 |
352 |
31.4 |
1.0 |
0.9 |
|
316 |
313 |
44.3 |
355 |
47.2 |
0.9 |
0.9 |
|
1000 |
349 |
45.7 |
394 |
13.2 |
1.0 |
1.0 |
|
2500 |
356 |
44.0 |
400 |
54.2 |
1.0 |
1.0 |
|
5000 |
294 |
17.6 |
369 |
11.1 |
0.9 |
1.0 |
|
A dest. |
- |
340 |
28.6 |
388 |
35.0 |
1.0 |
1.0 |
MMS |
1.3 (mg/plate) |
1342 |
62.6 |
/ |
/ |
3.9 |
/ |
2-AA |
10 |
/ |
/ |
972 |
62.1 |
/ |
2.5 |
SD = standard deviation; P = precipitation; B = background lawn reduced; N = no background lawn; C contamination
Mutation factor = mean revertants (test item) / mean revertants (vehicle control)
Table 3: Experiment II Results (Pre-incubation Test)
Treatment |
Dose (µg/plate) |
Revertant colonies per plate |
Mutation factor |
|||||
Without S9 (mean) |
SD |
With S9 (mean) |
SD |
-S9 |
+S9 |
|||
TA 98 |
||||||||
Test item |
10 |
23 |
6.4 |
20 |
0.0 |
0.9 |
0.7 |
|
31.6 |
21 |
1.2 |
29 |
8.7 |
0.9 |
1.0 |
||
100 |
25 |
9.1 |
26 |
7.2 |
1.0 |
1.0 |
||
316 |
28 |
4.4 |
25 |
2.1 |
1.1 |
0.9 |
||
1000 |
23 |
6.1 |
25 |
2.3 |
0.9 |
0.9 |
||
2500 |
18 |
2.1 |
26 |
4.5 |
0.7 |
1.0 |
||
5000 |
5 [B] |
2.5 |
13 [B] |
3.1 |
0.2 |
0.5 |
||
A dest. |
- |
25 |
6.0 |
27 |
4.5 |
1.0 |
1.0 |
|
4-NOPD |
10 |
441 |
94.5 |
/ |
/ |
17.9 |
/ |
|
2-AA |
2.5 |
/ |
|
1027 |
|
/ |
37.6 |
|
TA 100 |
||||||||
Test item |
10 |
83 |
11.0 |
79 |
6.1 |
0.9 |
0.8 |
|
31.6 |
95 |
4.9 |
89 |
5.1 |
1.0 |
0.9 |
||
100 |
94 |
16.0 |
81 |
6.4 |
1.0 |
0.9 |
||
316 |
92 |
10.1 |
93 |
9.0 |
1.0 |
1.0 |
||
1000 |
81 |
8.5 |
72 |
3.2 |
0.9 |
0.8 |
||
2500 |
42 [B] |
4.9 |
68 |
7.8 |
0.4 |
0.7 |
||
5000 |
20 [B] |
16.1 |
48 [B] |
18.4 |
0.2 |
0.5 |
||
A dest. |
- |
94 |
3.157.8 |
95 |
13.1 |
1.0 |
1.0 |
|
NaN3 |
10 |
587 |
57.8 |
/ |
/ |
6.3 |
/ |
|
2-AA |
2.5 |
/ |
/ |
814 |
142.8 |
/ |
8.6 |
|
TA 1535 |
||||||||
Test item |
10 |
22 |
0.6 |
13 |
2.0 |
1.1 |
0.8 |
|
31.6 |
22 |
2.3 |
14 |
0.6 |
1.2 |
0.9 |
||
100 |
20 |
2.1 |
14 |
2.0 |
1.0 |
0.9 |
||
316 |
19 |
2.1 |
15 |
0.6 |
1.0 |
0.9 |
||
1000 |
23 |
3.2 |
15 |
2.1 |
1.2 |
0.9 |
||
2500 |
3 [B] |
1.5 |
4 [B] |
1.0 |
0.1 |
0.3 |
||
5000 |
2 [B] |
0.6 |
1 [B] |
0.6 |
0.1 |
0.1 |
||
A dest. |
- |
19 |
1.7 |
16 |
|
1.0 |
1.0 |
|
NaN3 |
10 |
250 |
13.1 |
/ |
/ |
13.2 |
/ |
|
2-AA |
2.5 |
/ |
/ |
684 |
85.9 |
/ |
42.8 |
|
TA 1537 |
||||||||
Test item |
10 |
11 |
1.2 |
8 |
1.0 |
0.9 |
1.0 |
|
31.6 |
13 |
1.5 |
8 |
1.5 |
1.1 |
1.0 |
||
100 |
12 |
0.6 |
8 |
2.3 |
1.0 |
1.0 |
||
316 |
12 |
2.6 |
8 |
0.6 |
1.0 |
1.0 |
||
1000 |
11 |
0.6 |
9 |
0.6 |
0.9 |
1.1 |
||
2500 |
5 [B] |
0.6 |
4 [B] |
3.1 |
0.4 |
0.5 |
||
5000 |
2 [B] |
1.0 |
0 [B] |
1.5 |
0.2 |
0.0 |
||
A dest. |
- |
12 |
2.0 |
8 |
0.6 |
1.0 |
1.0 |
|
4-NOPD |
40 |
111 |
8.6 |
/ |
/ |
9.3 |
/ |
|
2-AA |
2.5 |
/ |
/ |
87 |
4.0 |
/ |
10.9 |
|
|
TA 102 |
|||||||
Test item |
10 |
268 |
5.5 |
359 |
39.2 |
1.0 |
0.9 |
|
31.6 |
281 |
23.9 |
379 |
12/1 |
1.0 |
1.0 |
||
100 |
262 |
17.4 |
359 |
6.7 |
1.0 |
0.9 |
||
316 |
243 |
14.0 |
329 |
31.0 |
0.9 |
0.8 |
||
1000 |
308 |
25.4 |
354 |
30.1 |
1.1 |
0.9 |
||
2500 |
308 |
16.3 |
391 |
21.0 |
1.1 |
1.0 |
||
5000 |
193 |
12.1 |
385 |
16.5 |
0.7 |
1.0 |
||
A dest. |
- |
275 |
27.5 |
389 |
2.5 |
1.0 |
1.0 |
|
MMS |
1.3 (mg/plate) |
744 |
78.4 |
/ |
/ |
2.7 |
/ |
|
2-AA |
10 |
/ |
/ |
866 |
95.6 |
/ |
2.2 |
|
SD = standard deviation; P = precipitation; B = background lawn reduced; N = no background lawn; C contamination
Mutation factor = mean revertants (test item) / mean revertants (vehicle control)
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.
All criteria of validity were met. The negative control plates (A. dest.) with and without S9 mix are within the historical control data range.The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.
In the main study cytotoxicity was noted in the experiments without and with metabolic activation at the top concentration of 2500 μg test item/mL medium.
Tests without metabolic activation (4- and 24-hour exposure)
The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with the test item at concentrations of 125, 250, 500, 1000, 1500, 2000 or 2500 μg test item/mL medium (4-h or 24-h exposure) in the absence of metabolic activation ranged from 1.0% to 3.5%. These results were within the range of the historical control data (0 - 4%).
The result for the vehicle control cultures was a mean of 1% cells with aberrations (excluding gaps), which is within the historical control range. The positive control cultures had a significantly increased frequency of cells with aberrations, which was in line with the historical control range. Therefore, the test is considered to be valid.
Test with metabolic activation (4-hour exposure)
The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with the test item at concentrations of 125, 250, 500, 1000, 1500, 2000 or 2500 μg test item/mL medium in the presence of metabolic activation in the first and second experiment ranged from 0.5% to 4.0%. These results were within the range of the historical control data (0 - 4%).
The result for the vehicle control cultures was a mean of 1% cells with aberrations (excluding gaps), which is within the historical control range. The positive control cultures had a significantly increased frequency of cells with aberrations, which was in line with the historical control range. Therefore, the test is considered to be valid.
Table 1. Estimation of cytotoxicity
Concentration (µg/mL medium) |
S9 mix |
Metaphases/1000 cells |
Miotic index MI#1 |
Miotic index MI % |
|
4 h exposure |
|||||
0 |
+ |
48 |
1.00 |
100 |
|
10 |
+ |
48 |
1.00 |
200 |
|
25 |
+ |
22 |
0.46 |
46 |
|
100 |
+ |
35 |
0.73 |
73 |
|
250 |
+ |
27 |
0.55 |
56 |
|
1000 |
+ |
60 |
1.25 |
125 |
|
2500#2 |
+ |
11 |
0.23 |
23 |
|
5000#2 |
+ |
20 |
0.42 |
42 |
|
24 h exposure |
|||||
0 |
- |
52 |
1.00 |
100 |
|
10 |
- |
51 |
0.98 |
98 |
|
25 |
- |
70 |
1.35 |
135 |
|
100 |
- |
48 |
0.92 |
92 |
|
250 |
- |
56 |
1.08 |
108 |
|
1000 |
- |
39 |
0.75 |
75 |
|
2500#2 |
- |
20 |
0.38 |
38 |
|
5000#2 |
- |
0 |
0.00 |
0 |
|
#1 = mitotic index (MI): number of metaphases/1000 cells; solvent control = 1.00
#2 = haemolysis
+ = with metabolic activation
- = without metabolic activation
Table 2. Experiments without metabolic activation (S9 mix)
1stexperiment, 4 h exposure |
2ndexperiment, 24 h exposure |
||||||||
Treatment (µg/mL medium) |
Miotic Index MI#1 |
MI % |
No. of metaphases scored |
% of cells with aberrations excluding gaps |
Treatment (µg/mL medium) |
Miotic Index MI#1 |
MI % |
No. of metaphases scored |
% of cells with aberrations excluding gaps |
Aqua ad iniectabilia |
|||||||||
0 |
1.00 |
100 |
200 |
1.0 |
0 |
1.00 |
100 |
200 |
1.0 |
Test item |
|||||||||
125 |
1.06 |
106 |
200 |
1.5 |
125 |
1.00 |
100 |
200 |
1.5 |
250 |
0.95 |
95 |
200 |
1.0 |
250 |
1.01 |
101 |
200 |
1.0 |
500 |
0.90 |
90 |
200 |
1.5 |
500 |
0.96 |
96 |
200 |
1.0 |
1000 |
0.87 |
87 |
200 |
1.5 |
1000 |
0.98 |
98 |
200 |
1.5 |
1500 |
1.01 |
101 |
200 |
1.5 |
1500 |
0.83 |
83 |
200 |
1.5 |
2000#2 |
0.93 |
93 |
200 |
2.5 |
2000#2 |
0.74 |
74 |
200 |
2.0 |
2500#2 |
0.31 |
31 |
200 |
3.5 |
2500#2 |
0.34 |
34 |
200 |
3.0 |
Mitomycin C |
|||||||||
0.2 |
0.81 |
81 |
200 |
10.5 s. |
0.2 |
0.74 |
74 |
200 |
9.5 s. |
s. = significantly different from negative control (p <= 0.05)
#1 – miotic index (MI): no. of metaphases/1000 cells: negative control = 1.00
#2 = haemolysis
Table 3. Experiments with metabolic activation (S9 mix)
1stexperiment, 4 h exposure |
2ndexperiment, 4 h exposure |
||||||||
Treatment (µg/mL medium) |
Miotic Index MI#1 |
MI % |
No. of metaphases scored |
% of cells with aberrations excluding gaps |
Treatment (µg/mL medium) |
Miotic Index MI#1 |
MI % |
No. of metaphases scored |
% of cells with aberrations excluding gaps |
Aqua ad iniectabilia |
|||||||||
0 |
1.00 |
100 |
200 |
1.0 |
0 |
1.00 |
100 |
200 |
1.0 |
Test item |
|||||||||
125 |
1.03 |
103 |
200 |
1.0 |
125 |
0.86 |
86 |
200 |
1.0 |
250 |
1.01 |
101 |
200 |
0.5 |
250 |
0.87 |
87 |
200 |
1.5 |
500 |
1.07 |
107 |
200 |
1.5 |
500 |
1.14 |
114 |
200 |
1.5 |
1000 |
1.20 |
120 |
200 |
1.5 |
1000 |
1.19 |
119 |
200 |
1.5 |
1500 |
1.03 |
103 |
200 |
1.5 |
1500 |
0.86 |
86 |
200 |
2.5 |
2000#2 |
1.15 |
115 |
200 |
3.0 |
2000#2 |
0.54 |
54 |
200 |
3.0 |
2500#2 |
0.28 |
028 |
200 |
4.0 |
2500#2 |
0.32 |
32 |
200 |
3.5 |
Cyclophosphamide |
|||||||||
10 |
1.23 |
123 |
200 |
8.0 s. |
20 |
0.66 |
66 |
200 |
8.5 s. |
s. = significantly different from negative control (p <= 0.05)
#1 – miotic index (MI): no. of metaphases/1000 cells: negative control = 1.00
#2 = haemolysis
Table 4. pH and osmality data of the negative control adn of all test item formulations in the medium
Concentration of test item [μg/mL medium] |
pH value |
osmolality [mOsmol/kg] |
Negative control |
7.51 |
280.5 |
125 |
7.51 |
276.0 |
250 |
7.51 |
285.5 |
500 |
7.52 |
295.0 |
1000 |
7.46 |
293.5 |
1500 |
7.50 |
293.5 |
2000 |
7.45 |
295.0 |
2500 |
7.64 |
299.0 |
Table 1. Results of trials 1 - 8
Concentration (µg/mL) |
CE |
RTG |
MC |
MF |
AVG MF |
Trial 1 (Without S9) |
|||||
DMSO 0 |
62 |
95 |
56 |
30 |
|
|
68 |
98 |
90 |
44 |
|
|
65 |
95 |
66 |
34 |
|
|
80 |
112 |
153 |
64 |
43 |
3.125 |
77 |
106 |
84 |
36 |
|
|
59 |
97 |
151 |
85 |
61 |
6.25 |
71 |
119 |
176 |
83 |
|
|
73 |
107 |
160 |
74 |
78 |
12.5 |
90 |
145 |
133 |
49 |
|
|
67 |
133 |
160 |
80 |
65 |
25 |
84 |
90 |
247 |
99 |
|
|
65 |
88 |
130 |
67 |
83 |
50 |
86 |
82 |
192 |
75 |
|
|
76 |
98 |
145 |
64 |
69 |
100 |
Lethal |
- |
- |
- |
- |
|
Lethal |
- |
- |
- |
- |
MMS 15 |
27 |
21 |
135 |
167 |
|
|
25 |
23 |
219 |
298 |
232 |
Concentration (µg/mL) |
CE |
RTG |
MC |
MF |
AVG MF |
Trial 2 (Without S9) |
|||||
DMSO 0 |
80 |
100 |
115 |
48 |
|
|
74 |
108 |
119 |
53 |
|
|
86 |
106 |
115 |
45 |
|
|
61 |
87 |
83 |
45 |
48 |
10 |
75 |
112 |
107 |
47 |
|
|
66 |
94 |
137 |
69 |
58 |
20 |
67 |
89 |
119 |
59 |
|
|
53 |
76 |
70 |
44 |
52 |
30 |
71 |
70 |
98 |
46 |
|
|
64 |
86 |
144 |
75 |
60 |
40 |
77 |
77 |
126 |
54 |
|
|
- |
- |
- |
- |
- |
50 |
94 |
61 |
191 |
68 |
|
|
68 |
56 |
163 |
80 |
74 |
60 |
81 |
27 |
363 |
150 |
|
|
77 |
16 |
595 |
256 |
203 |
70 |
Lethal |
- |
- |
- |
- |
|
Lethal |
- |
- |
- |
- |
MMS 15 |
34 |
30 |
683 |
666 |
|
|
29 |
27 |
573 |
662 |
664 |
Concentration (µg/mL) |
CE |
RTG |
MC |
MF |
AVG MF |
Trial 3 (Without S9) |
|||||
DMSO 0 |
76 |
103 |
84 |
37 |
|
|
71 |
104 |
73 |
34 |
|
|
82 |
97 |
98 |
40 |
|
|
74 |
96 |
58 |
26 |
34 |
50 |
60 |
71 |
65 |
36 |
|
|
75 |
75 |
82 |
36 |
36 |
55 |
68 |
44 |
68 |
33 |
|
|
53 |
71 |
69 |
44 |
38 |
60 |
72 |
63 |
107 |
50 |
|
|
72 |
74 |
134 |
62 |
56 |
65 |
64 |
71 |
87 |
45 |
|
|
79 |
68 |
93 |
39 |
42 |
70 |
80 |
83 |
107 |
44 |
|
|
- |
- |
- |
- |
- |
MMS 15 |
36 |
26 |
127 |
119 |
|
|
26 |
23 |
156 |
197 |
158 |
Concentration (µg/mL) |
CE |
RTG |
MC |
MF |
AVG MF |
Trial 4 (Without S9) |
|||||
DMSO 0 |
103 |
115 |
113 |
36 |
|
|
108 |
149 |
112 |
35 |
|
|
99 |
74 |
62 |
21 |
|
|
93 |
62 |
158 |
57 |
37 |
30 |
94 |
109 |
72 |
26 |
|
|
96 |
94 |
107 |
37 |
31 |
40 |
103 |
72 |
137 |
44 |
|
|
104 |
86 |
119 |
38 |
41 |
50 |
94 |
61 |
107 |
38 |
|
|
104 |
77 |
117 |
37 |
38 |
60 |
95 |
78 |
134 |
47 |
|
|
121 |
93 |
148 |
41 |
44 |
70 |
107 |
33 |
144 |
45 |
|
|
121 |
24 |
133 |
37 |
41 |
80 |
Lethal |
- |
- |
- |
- |
|
Lethal |
- |
- |
- |
- |
MMS 15 |
72 |
18 |
478 |
222 |
|
|
45 |
25 |
366 |
273 |
248 |
Concentration (µg/mL) |
CE |
RTG |
MC |
MF |
AVG MF |
Trial 5 (Without S9) |
|||||
DMSO 0 |
63 |
104 |
169 |
90 |
|
|
75 |
105 |
198 |
88 |
|
|
58 |
102 |
120 |
69 |
|
|
71 |
89 |
158 |
75 |
80 |
50 |
70 |
91 |
101 |
48 |
|
|
56 |
112 |
155 |
92 |
70 |
55 |
74 |
72 |
99 |
44 |
|
|
70 |
84 |
88 |
42 |
43 |
60 |
82 |
60 |
127 |
52 |
|
|
74 |
75 |
98 |
44 |
48 |
65 |
67 |
21 |
124 |
61 |
|
|
98 |
20 |
171 |
58 |
60 |
70 |
Lethal |
- |
- |
- |
|
|
Lethal |
- |
- |
- |
|
MMS 15 |
26 |
23 |
327 |
414 |
|
|
26 |
24 |
321 |
412 |
413 |
Concentration (µg/mL) |
CE |
RTG |
MC |
MF |
AVG MF |
Trial 6 (With S9) |
|||||
DMSO 0 |
77 |
103 |
74 |
32 |
|
|
70 |
88 |
53 |
25 |
|
|
64 |
109 |
61 |
32 |
30 |
50 |
48 |
82 |
47 |
33 |
|
|
37 |
52 |
64 |
57 |
45 |
60 |
52 |
68 |
75 |
49 |
|
|
40 |
61 |
44 |
36 |
42 |
70 |
62 |
78 |
48 |
26 |
|
|
21 |
28 |
35 |
56 |
41 |
80 |
46 |
42 |
35 |
25 |
|
|
- |
- |
- |
- |
- |
90 |
58 |
7 |
26 |
15 |
|
|
27 |
3 |
35 |
43 |
29 |
MCA 2.5 |
32 |
40 |
466 |
483 |
|
|
16 |
18 |
430 |
915 |
699 |
Concentration (µg/mL) |
CE |
RTG |
MC |
MF |
AVG MF |
Trial 7 (With S9) |
|||||
DMSO 0 |
104 |
110 |
84 |
27 |
|
|
87 |
97 |
35 |
13 |
|
|
90 |
94 |
69 |
26 |
22 |
55 |
89 |
98 |
63 |
24 |
|
|
- |
- |
- |
- |
- |
65 |
92 |
52 |
63 |
24 |
|
|
91 |
63 |
83 |
30 |
27 |
75 |
89 |
45 |
47 |
17 |
|
|
97 |
41 |
95 |
33 |
25 |
85 |
75 |
7 |
36 |
16 |
|
|
91 |
19 |
75 |
28 |
22 |
95 |
Lethal |
- |
- |
- |
- |
|
Lethal |
- |
- |
- |
- |
MCA 2.5 |
57 |
39 |
527 |
307 |
|
|
69 |
44 |
512 |
249 |
278 |
Concentration (µg/mL) |
CE |
RTG |
MC |
MF |
AVG MF |
Trial 8 (With S9) |
|||||
DMSO 0 |
103 |
102 |
104 |
34 |
|
|
92 |
109 |
168 |
61 |
|
|
84 |
92 |
66 |
26 |
|
|
90 |
96 |
62 |
23 |
36 |
60 |
112 |
94 |
118 |
35 |
|
|
108 |
83 |
120 |
37 |
36 |
65 |
76 |
68 |
57 |
25 |
|
CE = cloning efficiency %; RTG = relative total growth %; MC = mutant colony count; MF = mutant fraction (mutant colonies per 10^6 clonable cells); AVG MF = avergage group mutant fraction; underline = p<5%
Eight acceptable experiments were conducted, five in the absence of S9 mix. In the first of these, statistically significant increases in mutant fraction were observed at three dose levels: 6.25,25,and 50 µg/ml; 100 µg/ml was a lethal concentration. Over the nonlethal range, there were generally elevated mutant fractions, the highest being 1.9-fold the control level at 25 µg/ml. Although these increases in mutant fraction were significant, the lack of an obvious dose-related response with a relatively soluble chemical over a dose range which was not toxic encourages speculation that the increases were not due to the test material.
In the second experiment without S9 mix, there was a clearly significant response at 60 µg/ml, but at no other dose. The RTG was about 22%. Trial 3 gave a statistically significant response (1.7-fold increase) at 60 µg/ml, but not at 65 µg/ml. The mutant fraction at 70 µg/ml was only 44/10^6 survivors, so this single culture result supports the view that the statistically significant result at the lower dose level was a chance event. The succeeding experiments without S9 mix were unambiguously negative, as were two experiments with S9 mix. The last experiment with S9 mix was inconclusive because the RTG at 80 µg/mL was about 86% and there was no indication of a mutagenic response.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Mode of Action Analysis / Human Relevance Framework
Not applicable as no adverse effects observed in the available studies.
Additional information
Bacteria reverse gene mutation assay
In accordance with OECD 471, the test item sodium octane-1-sulphonate was tested for its potential to induce gene mutations. A plate incorporation test and pre-incubation test was conducted with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.
In three independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate.
The following concentrations referring to the main constituent of the test item were prepared and used in the experiments:
Pre-Experiment (Part of Experiment I):
2.53, 8.00, 25.3, 80.0, 253, 800, 2000 and 4000 µg/plate (TA 98 and TA 100)
Experiment I:
10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (TA 1535, TA 1537 and TA 102)
Experiment II:
10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (TA 98, TA 100, TA 1535, TA 1537 and TA 102)
These concentrations correspond to the following concentrations of the test item:
Pre-Experiment (Part of Experiment I):
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (TA 98 and TA 100)
Experiment I:
12.5, 39.5, 125, 395, 1250, 3125 and 6250 µg/plate (TA 1535, TA 1537 and TA 102)
Experiment II:
12.5, 39.5, 125, 395, 1250, 3125 and 6250 µg/plate (TA 98, TA 100, TA 1535, TA 1537 and TA 102).
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
No toxic effects of the test item were noted in tester strain TA 102 up to the highest dose group evaluated (with and without metabolic activation) in experiment I and II.
Toxic effects of the test item were noted in all other tester strains evaluated in the pre-experiment, experiment I and II.
In the pre-experiment toxic effects of the test item were observed in tester strain TA 98 at concentrations of 2000 μg/plate and higher (without metabolic activation) and at a concentration of 4000 μg/plate (with metabolic activation). In tester strain TA 100 toxic effects of the test item were noted at concentrations of 800 μg/plate and higher (without metabolic activation) and at a concentration of 4000 μg/plate (with metabolic activation).
In experiment I toxic effects of the test item were seen in tester strain TA 1535 at a concentration of 5000 μg/plate (without metabolic activation). In tester strain TA 1537 toxic effects of the test item were observed at a concentration of 5000 μg/plate (with and without metabolic activation).
In experiment II toxic effects of the test item were noted in tester strain TA 98 at a concentration of 5000 μg/plate (with and without metabolic activation). In tester strain TA 100 toxic effects of the test item were seen at concentrations of 2500 μg/plate and higher (without metabolic activation) and at a concentration of 5000 μg/plate (with metabolic activation). In tester strains TA 1535 and TA 1537 toxic effects of the test item were observed at concentrations of 2500 μg/plate and higher (with and without metabolic activation).
No biologically relevant increases in revertant colony numbers in any of the five tester strains were observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. All criteria of validity were met. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.
In vitro chromosome aberration test
A test according to OECD Guideline 473 was conducted in order to determine whether the test item caused structural chromosomal aberrations in cultured mammalian cells.
Samples of the test item were assayed in an in vitro cytogenetic study using human lymphocyte cultures both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced animals.
The test was carried out employing two exposure times without S9 mix: 4 and 24 hours, and one exposure time with S9 mix: 4 hours. The experiment with S9 mix was carried out twice. The harvesting time was 24 hours after starting of exposure. The incubation procedure took place in the dark. The study was conducted in duplicate.
The test item was completely dissolved in aqua ad iniectabilia. A correction factor of 1.02 was used as the purity of test item was 98% only. The vehicle served as the negative control.
Preliminary cytotoxicity study
A preliminary cytotoxicity study noted cytotoxicity at a concentration of 2500 μg test item/mL with and without metabolic activation (4-h or 24-h exposure). The highest concentration employed in the main study was 2500 test item/mL medium without and with metabolic activation.
Mitomycin C and cyclophosphamide were employed as positive controls in the absence and presence of metabolic activation, respectively.
Tests without metabolic activation (4- and 24-hour exposure)
The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with test item at concentrations of 125, 250, 500, 1000, 1500, 2000 or 2500 μg test item/mL medium (4-h or 24-h exposure) in the absence of metabolic activation ranged from 1.0% to 3.5%. These results were within the range of the historical control data (0 - 4%).
The result for the vehicle control cultures was a mean of 1% cells with aberrations (excluding gaps), which is within the historical control range. The positive control cultures had a significantly increased frequency of cells with aberrations, in line with the historical control range. Therefore, the test is considered to be valid.
Test with metabolic activation (4-hour exposure)
The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with test item at concentrations of 125, 250, 500, 1000, 1500, 2000 or 2500 μg test item /mL medium in the presence of metabolic activation in the first and second experiment ranged from 0.5% to 4.0%. These results were within the range of the historical control data (0 - 4%).
The result for the vehicle control cultures was a mean of 1% cells with aberrations (excluding gaps), which is within the historical control range. The positive control cultures had a significantly increased frequency of cells with aberrations, in line with the historical control range. Therefore, the test is considered to be valid. No test item-related polyploidy or endoreduplication were noted in the experiments without or with metabolic activation.
Under the conditions of the study, tested up to a cytotoxic concentration of 2500 μg test item/mL medium, in the absence and in the presence of metabolic activation, employing two exposure times (without S9) and one exposure time (with S9), revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage.
In the same test, Mitomycin C and cyclophosphamide induced significant increases in the frequency of damaged cells, which confirmed the validity of this assay.
In vitro mammalian cell gene mutation test
A mammalian cell gene mutation assay using mouse lymphoma L5178Y cells, in a test that was similar to an OECD 476 In Vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes (latest version adopted 29 July 2016), was performed using the test substance sodium dodecyl sulphate (read-across source substance for target substance: sodium octane-1-sulphonate monohydrate).
The test item was tested in both the absence and presence of metabolic activation (S9 mix) at concentrations of: -S9: 3.125, 6.25, 10, 12.5, 20, 25, 30, 40, 50, 55, 60, 65, 70, 80 and 100 μg/mL, +S9: 50, 55, 60, 65, 70, 75, 80, 85, 90 and 95 μg/mL.
Eight acceptable experiments were conducted, five in the absence of S9 mix. In the first of these, statistically significant increases in mutant fraction were observed at three dose levels: 6.25, 25, and 50 µg/ml; 100 µg/ml was a lethal concentration. Over the nonlethal range, there were generally elevated mutant fractions, the highest being 1.9-fold the control level at 25 µg/ml. Although these increases in mutant fraction were significant, the lack of an obvious dose-related response with a relatively soluble chemical over a dose range which was not toxic encourages speculation that the increases were not due to the test material.
In the second experiment without S9 mix, there was a clearly significant response at 60 µg/ml, but at no other dose. The RTG was about 22%. Experiment 3 gave a statistically significant response (1.7-fold increase) at 60 µg/ml, but not at 65 µg/ml. The mutant fraction at 70 µg/ml was only 44/10^6 survivors, so this single culture result supports the view that the statistically significant result at the lower dose level was a chance event. The succeeding experiments without S9 mix were unambiguously negative, as were two experiments with S9 mix. The last experiment with S9 mix was inconclusive because the RTG at 80 µg/mL was about 86% and there was no indication of a mutagenic response.
Although the higher dose levels of the test item induced toxic effects in the cells, there was no enhanced mutation rate in the treated or untreated cells, either with or without metabolic activation. The positive controls induced the expected mutagenic response and therefore the test was considered to be valid. Therefore, the test substance was considered to be non-mutagenic.
Justification for classification or non-classification
The substance does not meet the criteria for classfication in accordance with GHS or Regulation (EC) No 1272/2008 (CLP).
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