Registration Dossier
Registration Dossier
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EC number: - | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
- in vitro gene mutation study in bacteria: OECD 471 - Negative
- in vitro cytogenicity / chromosome aberration study in mammalian cells: OECD 473 - Negative
- in vitro gene mutation study in mammalian cells: OECD 490 - Negative
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2014-01-21 to 2014-08-12
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: International Conference on Harmonisation (ICH) Guidelines S2A and S2B
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- Test Material: Santicizer Platinum P-1400
Lot no: 644878
Supplier: Ferro Corporation (Bridgeport, NJ)
Manufactured date: 25 Semptember 2013
The sample was received at ambient conditions and stored at room temperature
(22 ± 3 °C). - Target gene:
- TA1537, TA98, TA100, TA1535, WP2 uvrA
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Details on mammalian cell type (if applicable):
- Rat liver S9 homogenate
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor™ 1254-induced rat liver S9 fraction
- Test concentrations with justification for top dose:
- In the confirmatory assay, Santicizer Platinum P-1400 was tested at 25, 50, 100, 250, 500, 1000, 2500 and 5000 µg/plate using the plate incorporation method.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: dimethylsulfoxide (DMSO)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 2-nitrofluorene
- sodium azide
- other: ICR-191 acridine, 2-aminoanthracene
- Details on test system and experimental conditions:
- For detection of potential- acting mutagens, the Aroclor 1254-induced rat liver S9 fraction was used. The metabolic activation mixture was prepared freshly everyday. The S9 mixture (7.5%v/v) consisted of:
0.75 mL of S9 fraction
0.20 mL of MgCl2 (0.4 M) - KCl (1.65 M)
0.05 mL of glucose-6-phosphate (1 M)
0.40 mL of nicotinamide adenine dinucleotide phosphate (NADP) (0.1 M)
5.00 mL of phosphate buffered saline
3.60 mL of sterile distilled deionized water - Evaluation criteria:
- Criteria for Acceptability and Interpretation of Assay
The vehicle and positive control substance plates for the current study were compared against revertant count ranges that are found in historical data ranges.
Criteria for Positive Response
The test substance was considered positive for mutagenicity if it induced an increase of revertants per plate with increasing concentration. The increases should be at least twotimes the vehicle control substance background frequency for strains with highspontaneous levels (i.e., TA100) and three times for those with low spontaneous levels(TA1537, TA98, TA1535 and WP2 uvrA). These increases should be seen in at least twoor more successive concentrations or the response should be repeatable at a single concentration.
Criteria for Negative Response
The test substance was considered to be negative for inducing mutagenicity if it did not induce a response which fulfills the criteria for a positive response.
Criteria for Equivocal Response
Cases which did not clearly fit into the positive or negative criteria may be judged equivocal. In these cases the Study Director, based on sound scientific judgment, may take additional factors into consideration in evaluating the test results. - Statistics:
- For each concentration level and for each condition, the mean revertant count and standard deviation (SD) were determined.
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5000 µg/plate in TA1535 without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- ≥250 µg/plate in TA1537 without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at ≥2500 µg/plate in TA100 with and without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Precipitates were observed at >=500 ug/plate in all strains without metabolic activation and at >=2500 ug/plate in TA100 with and without metabolic activation.
- Remarks on result:
- other: strain/cell type: Aroclor 1254-induced rat liver S9 fraction
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results: negative
The test material Santicizer Platinum P1400 is considered to be negative for inducing mutagenicity under the condition of this study. - Executive summary:
The test material Santicizer Platinum P1400 was evaluated for mutagenic activity in the vistro Salmonella-E coli/mammalian microsome reverse assay using the plate incorporation method. In the initial assay, Santicizer Platinum P-1400 was tested at 25, 50, 100, 250, 500, 1000, 2500 and 5000 µg/plate using the plate incorporation method. Precipitates were observed at ≥500 µg/plate in all strains without metabolic activation and at ≥2500 µg/plate in all strains with metabolic activation. Cytotoxicity was observed at ≥1000 µg/plate in TA100 with and without metabolic activation.
In the confirmatory assay, Santicizer Platinum P-1400 was tested at 25, 50, 100, 250,500, 1000, 2500 and 5000 µg/plate using the plate incorporation method. Precipitates were observed at ≥500 µg/plate in all strains without metabolic activation and at ≥2500 µg/plate in all strains with metabolic activation. Cytotoxicity was observed at ≥250 µg/plate in TA1537 without metabolic activation, at ≥2500 µg/plate in TA100 with and without metabolic activation, and at 5000 µg/plate in TA1535 without metabolic activation.
In both assays, criteria for a negative response were met for all tester strains with and without metabolic activation. In conclusion, the test material Santicizer Platinum P1400 is considered to be negative for inducing mutagenicity under the condition of this study.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2015-11-25 to 2016-01-19
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- 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:
- Identification: 1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2- (phenylmethyl) ester
CAS No: 1200806-67-2
Physical state/Appearance: Clear colorless liquid
Batch: 0900
Purity: 98.8% †
Expiry Date: 22 October 2016
Storage Conditions: Room temperature in the dark
No correction for purity was made. - Species / strain / cell type:
- lymphocytes: human lymphocytes
- Details on mammalian cell type (if applicable):
- For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years inhouse data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
The details of the donors used were:
Preliminary Toxicity Test: male, aged 29 years
Main Experiment: male, aged 28 years
Cell Culture
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA). - Metabolic activation:
- with and without
- Metabolic activation system:
- S9 Microsomal fractions were prepared in-house from male rats induced with Phenobarbitone/β-Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4. The S9 homogenate was produced by homogenizing the liver in a 0.15M KCl solution (1g liver to 3 mL KCl) followed by centrifugation at 9000 g. The protein content of the resultant supernatant was adjusted to 20 mg/mL. Aliquots of the supernatant were frozen and stored at approximately -196 °C. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.
The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%. - Test concentrations with justification for top dose:
- The dose range for the Preliminary Cytotoxicity Test was 7.81 to 2000 µg/mL. The maximum dose was equivalent to a 2 mM concentration.
The dose levels of the controls and the test item are:
- 4(20)-hour without S9: 0*, 3.75, 7.5, 15*, 30*, 45*, 60*, 120, MMC 0.2, MMC 0.4*
- 4(20)-hour with S9 (2%): 0*, 7.5, 15, 30, 60*, 75*, 120*, 240*, CP 1.25*, CP 2.5
- 24-hour without S9: 0*, 7.5, 15*, 30*, 60*, 75*, 120, 240, MMC 0.1*, MMC 0.2
** = Dose levels selected for metaphase analysis
MMC = Mitomycin C
CP = Cyclophosphamide - Vehicle / solvent:
- DMSO
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES
buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin,
amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2
in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to
divide by the addition of phytohaemagglutinin (PHA).
DURATION
- Preincubation period:
CULTURE CONDITIONS
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood
With Metabolic Activation (S9) Treatment
After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.1 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1mL of 20% S9¯mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Cytotoxicity Test and Main Experiment. After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 ºC in 5% CO2 in humidified air.
Without Metabolic Activation (S9) Treatment
For the 4(20)-hour exposure in the absence of S9 - After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then re-suspended in the required volume of fresh MEM (including serum) and dosed with 0.1 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL. After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours. For the 24-hour exposure in the absence of S9, the exposure was continuous. Therefore, when the cultures were established the culture volume was a nominal 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 ºC, 5% CO2 in humidified air for 24 hours. The preliminary cytotoxicity test was performed using all three of the exposure conditions as described for the Main Experiment but with only single cultures.
- Preliminary test:
i) 4-hour exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
iii) 24-hour continuous exposure to the test item without S9-mix.
The dose range of test item used was 0, 7.5, 15, 30, 60, 75, 120 and 240 µg/mL.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test item cytotoxicity and for selection of the dose levels for the main test.
- Main Experiment
Three exposure groups were used for the Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 3.75, 7.5, 15, 30, 45, 60 and 120 µg/mL.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 7.5, 15, 30, 60, 75, 120 and 240 µg/mL.
iii) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest.
The dose range of test item used was 0, 7.5, 15, 30, 60, 75, 120 and 240 µg/mL.
- Cell Harvest
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/mL) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.
- Preparation of Metaphase Spreads
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labeled with the appropriate identification data.
- Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.
QUALITATIVE SLIDE ASSESSMENT:
To determine the quality of the metaphases and also
the cytotoxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.
STAINING
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.
CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
DETERMINATION OF CYTOTOXICITY
Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate). Where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985). - Evaluation criteria:
- The following criteria were used to determine a valid assay:
• The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range.
• All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations were analyzed. - Statistics:
- The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis. - Key result
- Species / strain:
- lymphocytes: human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- Not clastogenic
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Preliminary Toxicity Test:
The dose range for the Preliminary Cytotoxicity Test was 7.81 to 2000 µg/mL. The maximum dose was equivalent to a 2 mM concentration.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, ≥ 125 µg/mL, in the 4(20)-hour exposure groups and ≥ 250 µg/mL in the continuous exposure group. Hemolysis was observed following exposure to the test item ≥ 62.5 µg/mL in the 4(20)-hour exposure group in the absence of metabolic activation (S9), ≥ 250 µg/mL in the 4(20)-hour exposure group in the presence of S9 and ≥ 500 µg/mL in the 24-hour continuous exposure group. Hemolysis is an indication of a cytotoxic response in erythrocytes but is not indicative of any genotoxic response in the lymphocytes.
Main study:
The dose levels of the controls and the test item are given in the table below:
- 4(20)-hour without S9 0*, 3.75, 7.5, 15*, 30*, 45*, 60*, 120, MMC 0.2, MMC 0.4*
- 4(20)-hour with S9 (2%) 0*, 7.5, 15, 30, 60*, 75*, 120*, 240*, CP 1.25*, CP 2.5
- 24-hour without S9 0*, 7.5, 15*, 30*, 60*, 75*, 120, 240, MMC 0.1*, MMC 0.2
The qualitative assessment of the slides determined that the cytotoxicity was similar to that observed in the Preliminary Cytotoxicity Test and that metaphases deemed suitable for scoring were present up to the maximum test item dose level in all three exposure groups.
Precipitate observations were performed at the end of exposure in the blood-free cultures and was noted ≥ 120 µg/mL in the exposure groups in the absence of S9. No precipitate was observed at the end of exposure in the presence of S9. No haemolysis was observed at the end of exposure. The assay was
considered valid as it met all of the criteria. - Remarks on result:
- other: Not clastogenic
- Conclusions:
- The test item was considered to be non-clastogenic to human lymphocytes in vitro.
- Executive summary:
The study was conducted to detect the structural chromosomal aberrations in cultured mammalian cells.
Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation. The dose levels used in the Main Experiment were selected using data from the preliminary cytotoxicity test where the results indicated that the maximum concentration should be based on cytotoxicity.
All the positive control items induced statistically significant increases in the frequency of cells with chromosomal aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The results indicated that the test item was cytotoxic but did not induce any statistically significant increases in the frequency of cells with chromosomal aberrations, using a dose range that included a dose level that exceeded 55±5% mitotic inhibition. The vehicle (dimethyl sulphoxide) controls had cells with chromosomal aberration frequencies within the range expected for normal human lymphocytes, therefore the study is considered valid.
According to the conditions of this study, the test item, 1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2-(phenylmethyl) ester, CAS 1200806-67-2 was considered to be non-clastogenic to human lymphocytes in vitro.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- L5178Y mouse lymphoma assay
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2015-11-23 to 2015-12-15
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Deviations:
- yes
- Remarks:
- The deviation do not affect the validity, integrity or the result of the study.
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Deviations:
- yes
- Remarks:
- The deviation do not affect the validity, integrity or the result of the study.
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- yes
- Remarks:
- The deviation do not affect the validity, integrity or the result of the study. The deviation do not affect the validity, integrity or the result of the study.
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Version / remarks:
- Kanpoan No. 287 - - Environment Protection Agency`
Eisei No. 127 - - Ministry of Health and Welfare
Heisei 09/10/31 Kikyoku No. 2 - - Ministry of International Trade & Industry - Deviations:
- yes
- Remarks:
- The deviation do not affect the validity, integrity or the result of the study.
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Specific details on test material used for the study:
- Identification: 1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2-
(phenylmethyl) ester, CAS 1200806-67-2
Physical State / Appearance: Clear colorless liquid
Batch: 0900
Purity: 98.8%
Expiry Date: 22 October 2016
Storage Conditions: Room temperature, in the dark - Target gene:
- Thymidine Kinase Locus Gene Mutation Assay
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- Cell Line
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.
Cell Culture
The stocks of cells were stored in liquid nitrogen at approximately -196°C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at 37°C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study. Tests on master stocks of cells showed absence of mycoplasma.
Cell Cleansing
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/mL), Hypoxanthine (15 µg/mL), Methotrexate (0.3 µg/mL) and Glycine (22.5 µg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium. - Additional strain / cell type characteristics:
- not specified
- Metabolic activation system:
- Lot No. PB/βNF S9 17/05/15 was used in this study, and was pre-prepared in-house (outside the confines of the study) following standard procedures. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.
The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%. - Test concentrations with justification for top dose:
- The dose range used in the preliminary toxicity test was 7.81 to 2000 µg/mL for all three of the exposure groups.
The dose range used in the main study were ranging from 4 to 64 µg/mL in the 4-hour exposure groups and from 0.5 to 32 in the 24-hour exposure group), vehicle and positive controls. - Vehicle / solvent:
- DMSO
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- Test Item Preparation
Following solubility checks performed in-house, the test item was accurately weighed and formulated in dimethyl sulfoxide (DMSO) prior to serial dilutions being prepared. The maximum recommended dose level of 2000 µg/mL was investigated initially. Since the completion of the experimental phase of the study, the purity has been clarified as 98.8%.
However, dose levels were limited by marked toxicity of the test item, therefore, the nominal dose levels given in the report are considered adequate and the study meets the criteria of the Guideline. There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al. 1991).
Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/mL using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 7.81 to 2000 µg/mL for all three of the exposure groups. Following the exposure period the cells were washed twice with R10 medium, resuspended in R20 medium, counted and then serially diluted to 2 x 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained. The cultures were incubated at 37°C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post exposure toxicity, and a comparison of each exposure SG value to the concurrent vehicle control was performed to derive a percentage Relative Suspension Growth (%RSG) value.
Results from the preliminary cytotoxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) For non-toxic test items the upper test item concentrations will be 10mM, 2 mg/mL or 2µL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCBs) the upper dose level may need to be higher and the maximum concentration will be 5 mg/mL.
ii) Precipitating dose levels will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point.
iii) In the absence of precipitate and if cytotoxicity occurs, the highest concentration should be that which results in a reduction of the Relative Total Growth (RTG) to approximately
10 to 20 % of survival. This optimum upper level of cytotoxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al 2002).
Mutagenicity Test
Seven days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10^6 cells/mL in 10 mL aliquots in R10 medium in sterile plastic vial for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 10^6 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at eight dose levels of the test item (4 to 64 µg/mL in the 4-hour exposure groups and 0.5 to 32 in the 24-hour exposure group), vehicle and positive controls. To each vial was added 2 mL of S9-mix if required, 0.2 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24 hour exposure group). The exposure vessels were incubated at 37 °C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.
Measurement of Survival, Viability and Mutant Frequency
At the end of the exposure period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10^5 cells/mL. The cultures were incubated at 37°C with 5% CO2 in air and subcultured every 24 hours for the two day expression period, by counting and dilution to 2 x 10^5 cells/mL, unless the mean cell count was less than 3x 10^5 cells/mL in which case all the cells were maintained. On Day 2 of the experiment, the cells were counted, diluted to 10^4 cells/mL and plated in 96 well microtitre plates (2000 cells/well) in selective medium containing 4 µg/mL 5 trifluorothymidine (TFT) for the evaluation of mutant frequency. Cells were also diluted to 10 cells/mL and plated (2 cells/well) in non-selective medium for viability assessment (%V).
The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post exposure toxicity during the expression period as a comparison to the vehicle control. When combined with the Viability (%V) data it provides a Relative Total Growth (RTG) value.
Plate scoring
Microtitre plates were scored using a magnifying mirror box after ten to twelve days incubation at 37 °C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item (Cole et al, 1990). Colonies were scored manually by eye using qualitative judgment. Large colonies were defined as those that covered approximately ¼ to ¾ of the surface of the well and were generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies were scored as small colonies. Small colonies were normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, (2.5 mg/mL in phosphate buffered saline (PBS)), was added to each well of the mutation plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies. - Rationale for test conditions:
- Please see box below
- Evaluation criteria:
- Please see box below
- Statistics:
- Calculation of Percentage Relative Suspension Growth (%RSG)
Calculation of Day 2 Viability (%V)
Calculation of Relative Total Growth (RTG)
Calculation of Mutation Frequency (MF) - Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- only in absence of metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Preliminary Toxicity Test: The dose range of the test item used in the preliminary toxicity test was 7.81 to2000 µg/mL
There was evidence of marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls in all three of the exposure groups, with the most marked reductions observed in the 24-hour exposure group. Precipitate of the test item was observed at and above 62.5 µg/mLin all three exposure groups. Test item induced cytotoxicity was used to designate dose levels in the 4-hour and 24-hour exposure in the absence of metabolic activation and the incidence of precipitate was used as the limiting factor in designating the dose levels in the 4-hour exposure in the presence of metabolic activation.
A summary of the results from the test is presented in Table 1.
Mutagenicity Test:
There was evidence of cytotoxicity following exposure to the test item in 4-hour and 24-hour exposure groups in the absence of metabolic activation only, as indicated by the %RSG and RTG values. There was evidence of moderate reductions in viability (%V) in both the 4-hour and 24-hour exposure groups in the absence of metabolic activation, indicating that
residual cytotoxicity had occurred. Optimum levels of cytotoxicity were achieved in the 4-hour and 24-hour exposures in the absence of metabolic activation. In the 4-hour exposure in the absence of metabolic activation the 64 µg/mL dose level was plated out but later excluded from statistical analysis due to excessive cytotoxicity. In the 24-hour exposure in the absence of metabolic activation the 32 µg/mL was not plated out for viability or 5-TFT resistance due to excessive cytotoxicity. In the 4-hour exposure in the presence of metabolic
activation there was evidence of slight toxicity in the upper dose levels, as indicated by the %RSG and RTG values. Optimum levels of cytotoxicity could not be achieved in this exposure group due to dose level selection based on precipitate. There was no evidence of marked reductions in viability (%V) in the presence of metabolic activation, indicating that residual cytotoxicity had not occurred. Acceptable levels of cytotoxicity were seen with the positive control substances in all three exposure groups.
The mutant frequency values in the vehicle control groups were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.
The test item induced small but statistically significant and dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell in the 4-hour and 24-hour exposure groups in the absence of metabolic activationonly, with the largest increases observed around optimum levels of cytotoxicity. However the GEF was not exceeded at any test item dose levels, including those that achieved optimum levels of toxicity (Moore et al. 2006). Therefore the increases were considered to be of no toxicological relevance. In the 4-hour exposure in the
presence of metabolic activation the test item did not induce any statistically significant or dose related (linear trend) increases in the mutant frequency x10^-6 per viable cell. Precipitate of the test item was only observed in the 4–hour exposure groups at 64 µg/mL.
In all three exposure groups at all concentrations of the test item, the colony sizes were predominantly large further supporting the negative result given.
A summary of the results from the test are presented in the Table 2 in the section 'Any other information on results incl. tables'. - Conclusions:
- The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.
- Executive summary:
The study was conducted to assess the mutagenicity potential of the test item 1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2-(phenylmethyl) ester, CAS 1200806-67-2. The test was done accoriding to the OECD Guidelines for Testing of Chemicals No. 490 "In Vitro Mammalian Cell Gene Mutation Tests using the Thymidine Kinase Gene, Method B17 of Commission Regulation (EC) No. 440/2008and the US EPA OPPTS 870.5300 Guideline. It is also aligned with the Japanise MITI/MHW Guideline.
In the main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (DMSO), and positive controls using 4 -hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 hour exposure group in the absence of metabolic activation. The dose range of the test item used in the main test was selected following the results of a preliminary toxicity study.
The maximum dose level used in the Mutagenicity Test was limited by test item induced toxicity in the absence of metabolic activation and by precipitate of the test item in the presence of metabolic activation. The mutant frequency values in the vehicle control groups were acceptable for the L5178Y cell line at the TK +/- locus. The positive controls induced marked increases in the mutant frequency, indicating the satisfactory performance of the test and of the activity of the metabolizing system. The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure
groups.
In conclusion, according to the results of this study, the test item 1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2-(phenylmethyl) ester, CAS 1200806-67-2 did not induce a toxicological statistically significant increase in the mutant frequency at the TK +/- locus in L5178Y cells.
Referenceopen allclose all
Table 1. Initial Assay: Summary Results of Plate Incorporation Experiment: Revertant Colonies per Plate - Mean (SD)a |
||||||
Treatment Group |
µg/plate |
TA1537 |
TA98 |
TA100 |
TA1535 |
WP2uvrA |
Without Metabolic Activation (-S9) |
||||||
Dimethylsulfoxide (DMSO) |
100 µL |
4 (2) |
13 (3) |
88 (13) |
10 (3) |
16 (5) |
ICR-191 Acridine (ICR) |
0.5 |
272 (31) |
|
|
|
|
2-Nitrofluorene (2-NF) |
2.5 |
|
516 (72) |
|
|
|
Sodium azide (SA) |
1.0 |
|
|
496 (24) |
355 (15) |
|
4-nitroquinoline-N-oxide (NQNO) |
2.0 |
|
|
|
|
2533 (77) |
|
||||||
Santicizer Platinum P-1400 |
25 |
5 (2) |
10 (3) |
78 (8) |
9 (7) |
14 (4) |
50 |
5 (1) |
15 (2) |
84 (14) |
7 (1) |
14 (5) |
|
100 |
3 (1) |
14 (2) |
75 (21) |
7 (3) |
17 (1) |
|
250 |
3 (1) |
13 (2) |
62b(4) |
3 (1) |
17 (1) |
|
500d |
5 (1) |
15 (8) |
58b (14) |
6 (5) |
13 (5) |
|
1000d |
2 (0) |
15 (3) |
--c-- |
6 (3) |
17 (1) |
|
2500d |
3 (3) |
14 (7) |
--c-- |
5 (3) |
12 (3) |
|
5000d |
6 (2) |
13 (10) |
--c-- |
8 (1) |
17 (6) |
|
With Metabolic Activation (+S9) |
||||||
Dimethylsulfoxide (DMSO) |
100 µL |
7 (3) |
16 (5) |
94 (18) |
11 (3) |
16 (2) |
2-Aminoanthracene (2AA) |
2.5 |
61 (11) |
1253 (100) |
496 (116) |
102 (14) |
|
2-Aminoanthracene (2AA) |
10.0 |
|
|
|
|
176 (129) |
|
||||||
Santicizer Platinum P-1400 |
25 |
10 (5) |
22 (2) |
86 (15) |
10 (6) |
19 (4) |
50 |
3 (2) |
18 (7) |
102 (25) |
6 (0) |
21 (5) |
|
100 |
6 (3) |
17 (6) |
86 (11) |
10 (2) |
24 (9) |
|
250 |
5 (3) |
21 (5) |
66b(3) |
9 (3) |
19 (3) |
|
500d |
3 (1) |
18 (3) |
75b(9) |
7 (4) |
17 (4) |
|
1000d |
3 (3) |
23 (12) |
--c-- |
6 (3) |
14 (4) |
|
2500d |
6 (3) |
20 (3) |
--c-- |
8 (3) |
18 (3) |
|
5000d |
4 (2) |
18 (6) |
--c-- |
6 (4) |
25 (7) |
|
a Calculated from triplicate plates
b Slightly reduced background lawn
c Cytotoxicity: Reduced background lawn, plates not counted
d Precipitates present
Table 2. Confirmatory Assay: Summary Results of Plate Incorporation Experiment: Revertant Colonies per Plate - Mean (SD)a |
||||||
Treatment Group |
µg/plate |
TA1537 |
TA98 |
TA100 |
TA1535 |
WP2uvrA |
Without Metabolic Activation (-S9) |
||||||
Dimethylsulfoxide (DMSO) |
100 µL |
5 (2) |
28 (5) |
98 (17) |
9 (5) |
25 (7) |
ICR-191 Acridine (ICR) |
0.5 |
284 (44) |
|
|
|
|
2-Nitrofluorene (2-NF) |
2.5 |
|
712 (74) |
|
|
|
Sodium azide (SA) |
1.0 |
|
|
610 (41) |
365 (18) |
|
4-nitroquinoline-N-oxide (NQNO) |
2.0 |
|
|
|
|
2202 (228) |
|
||||||
Santicizer Platinum P-1400 |
25 |
3 (2) |
26 (4) |
79 (6) |
8 (3) |
27 94) |
50 |
9 (2) |
24 (8) |
70 (11) |
5 (2) |
30 (7) |
|
100 |
6(3) |
20 (1) |
72 (3) |
10 (1) |
30 (8) |
|
250 |
--c-- |
21 (3) |
64 (2) |
10 (5) |
34 (11) |
|
500e |
--c-- |
25 (6) |
69b(1) |
9 (2) |
29 (6) |
|
1000e |
--c-- |
24 (10) |
69b(7) |
9 (3) |
30 (6) |
|
2500e |
--c-- |
17 (6) |
--c-- |
8 (5) |
35 (7) |
|
5000e |
--c-- |
28 (16) |
--c-- |
4d(1) |
35 (10) |
|
With Metabolic Activation (+S9) |
||||||
Dimethylsulfoxide (DMSO) |
100 µL |
6 (1) |
25 (3) |
92 (11) |
12 (3) |
27 (10) |
2-Aminoanthracene (2AA) |
2.5 |
55 (14) |
1243 (52) |
695 (129) |
72 (9) |
|
2-Aminoanthracene (2AA) |
10.0 |
|
|
|
|
160 (21) |
|
||||||
Santicizer Platinum P-1400 |
25 |
5 (3) |
34 (4) |
82 (10) |
7 (1) |
25 (2) |
50 |
8 (2) |
18 (1) |
84 (8) |
10 (5) |
21 (10) |
|
100 |
6 (1) |
29 (5) |
84 (12) |
9 (3) |
30 (0) |
|
250 |
5 (2) |
18 (9) |
91 (15) |
4 (2) |
26 (8) |
|
500 |
7 (2) |
28 (4) |
74b(6) |
7 94) |
32 (7) |
|
1000 |
4 (4) |
21 (0) |
85b(10) |
7 (1) |
25 (2) |
|
2500e |
5 (2) |
34 (13) |
--c-- |
9 (2) |
32 (5) |
|
5000e |
8 (6) |
32 (4) |
--c-- |
6 (1) |
31 (3) |
|
a Calculated from triplicate plates
b Slightly reduced background lawn
c Cytotoxicity: Reduced background lawn, plates not counted
d Cytotoxicity: >50% reduction in mean number of revertant colonies
e Precipitates present
Table 2. Mitotic Index - Preliminary Toxicity Test |
||||||
Dose Level (µg/mL) |
4(20)-Hour Without S9 |
4(20)-Hour With S9 |
24-Hour Without S9 |
|||
Mitotic Index |
% of Control |
Mitotic Index |
% of Control |
Mitotic Index |
% of Control |
|
0 |
2.00 |
100 |
4.25 |
100 |
6.05 |
100 |
7.81 |
2.50 |
125 |
- |
- |
- |
- |
15.63 |
2.55 |
128 |
5.05 |
119 |
6.30 |
104 |
31.25 |
1.90 |
95 |
4.00 |
94 |
5.15 |
85 |
62.5 |
0.50H |
25 |
3.15 |
74 |
4.15 |
69 |
125 |
NM P H |
- |
2.20P |
52 |
1.00 |
17 |
250 |
NM P H |
- |
NM P H |
- |
NM P |
- |
500 |
NM P H |
- |
NM P H |
- |
NM P H |
- |
1000 |
NM P H |
- |
NM P H |
- |
NM P H |
- |
2000 |
NM P H |
- |
NM P H |
- |
NM P H |
- |
- = Not assessed for mitotic index
NM = No metaphases or insufficient metaphases suitable for scoring
P = Precipitate observed at end of exposure period in blood-free cultures
H = Haemolysis observed at the end of exposure in blood cultures
Table 3. Mitotic Index – Main Experiment (4(20)-hour Exposure Groups) |
||||
Dose Level (µg/mL) |
4(20)-Hour Without S9 |
4(20)-Hour With S9 |
||
Mean |
% of Control |
Mean |
% of Control |
|
0 |
4.68 |
100 |
5.33 |
100 |
3.75 |
- |
- |
NA |
NA |
7.5 |
6.58 |
141 |
- |
- |
15 |
6.58 |
141 |
- |
- |
30 |
4.60 |
98 |
4.55 |
85 |
45 |
3.23 |
60 |
NA |
NA |
60 |
1.60 |
34 |
3.90 |
73 |
75 |
NA |
NA |
4.40 |
83 |
120 |
- |
- |
3.65 |
69 |
240 |
NA |
NA |
1.73 |
32 |
Mitomycin C (MMC) 0.2 |
3.03 |
65 |
NA |
NA |
Mitomycin C (MMC) 0.4 |
1.55 |
33 |
NA |
NA |
Cyclophosphamide (CP) 1.25 |
NA |
NA |
1.98 |
37 |
Cyclophosphamide (CP) 2.5 |
NA |
NA |
0.53 |
10 |
P = Precipitate
NA = Not applicable
- = Not assessed for mitotic index
NM = No or too few metaphases present suitable for scoring
Table 4. Mitotic Index – Main Experiment (24-hour Exposure Group) |
||
Dose Level (µg/mL) |
24-Hour Without S9 |
|
Mean |
% of Control |
|
0 |
6.55 |
100 |
7.5 |
- |
- |
15 |
4.35 |
66 |
30 |
3.40 |
52 |
60 |
3.03 |
46 |
75 |
2.58 |
39 |
120 |
- |
- |
240 |
- |
- |
Mitomycin C (MMC) 0.1 |
2.50 |
38 |
Mitomycin C (MMC) 0.2 |
1.60 |
24 |
MMC = Mitomycin C
P = Precipitate
- = Not assessed for mitotic index
NM = No or too few metaphases present suitable for scoring
Table 5. Chromosome Aberration Test – Main Experiment 4(20)-hour Exposure Without Metabolic Activation (-S9) |
|||||||||||||
Treatment Group |
Replicate |
Mitotic Index (%) |
Number of cells scored |
Number of Aberrations |
Total Number of Aberrations |
Frequency of Aberrant Cells (%) |
|||||||
Gaps |
Chromatid |
Chromosome |
Others X |
(+Gaps) |
(-Gaps) |
(+Gaps) |
(- Gaps) |
||||||
Break |
Exchanges |
Breaks |
Exchanges |
||||||||||
Vehicle Control (DMSO) |
A |
5.95 |
150 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
1 |
1 |
1 |
B |
3.40 |
150 |
2 |
0 |
0 |
0 |
0 |
0 |
2 |
0 |
2 |
0 |
|
Total |
(100) |
300 |
2 |
1 |
0 |
0 |
0 |
0 |
3 |
1 |
3 (1.0) |
1 (0.3) |
|
15 µg/mL |
A |
6.55 |
150 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
1 |
1 |
1 |
B |
6.60 |
150 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
1 |
1 |
1 |
|
Total |
(141) |
300 |
0 |
2 |
0 |
0 |
0 |
0 |
2 |
2 |
2 (0.7) |
2 (0.7) |
|
30 µg/mL |
A |
4.95 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
4.25 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Total |
(98) |
300 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 (0.0) |
0 (0.0) |
|
45 µg/mL |
A |
3.25 |
150 |
1 |
0 |
0 |
0 |
1 |
0 |
2 |
1 |
2 |
1 |
B |
3.20 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Total |
(69) |
300 |
1 |
0 |
0 |
0 |
1 |
0 |
2 |
1 |
2 (0.7) |
1 (0.3) |
|
60 µg/mL |
A |
1.30 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
1.90 |
150 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
1 |
1 |
1 |
|
Total |
(34) |
300 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
1 |
1 (0.3) |
1 (0.3) |
|
Positive Control Mitomycin C (MMC) 0.4 |
A |
1.55 |
50a |
1 |
12 |
8 |
1 |
0 |
0 |
22 |
21 |
16 |
16 |
B |
1.55 |
52a |
1 |
9 |
7 |
4 |
0 |
0 |
21 |
20 |
17 |
16 |
|
Total |
33 |
102 |
2 |
21 |
15 |
5 |
0 |
0 |
43 |
41 |
33 (32.4) |
32*** (31.4) |
|
aSlide evaluation terminated when 15 cells with aberrations (excluding gaps) had been observed
*** P < 0.001
DMSO Dimethyl sulphoxide
Table 6. Chromosome Aberration Test – Main Experiment 4(20)-hour Exposure With Metabolic Activation (2% S9) |
|||||||||||||
Treatment Group |
Replicate |
Mitotic Index (%) |
Number of cells scored |
Number of Aberrations |
Total Number of Aberrations |
Frequency of Aberrant Cells (%) |
|||||||
Gaps |
Chromatid |
Chromosome |
Others X |
(+Gaps) |
(-Gaps) |
(+Gaps) |
(- Gaps) |
||||||
Break |
Exchanges |
Breaks |
Exchanges |
||||||||||
Vehicle Control (DMSO) |
A |
6.20 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
4.45 |
150 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
1 |
1 |
1 |
|
Total |
(100) |
300 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
1 |
1 (0.30) |
1 (0.3) |
|
60 µg/mL |
A |
4.90 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
2.90 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Total |
(73) |
300 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 (0.0) |
0 (0.0) |
|
75 µg/mL |
A |
3.75 |
150 |
1 |
2 |
0 |
1 |
0 |
0 |
4 |
3 |
4 |
3 |
B |
5.05 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Total |
(83) |
300 |
1 |
2 |
0 |
1 |
0 |
0 |
4 |
3 |
4 (1.3) |
3 (1.0) |
|
120 µg/mL |
A |
3.65 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
3.65 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Total |
(69) |
300 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 (0.0) |
0 (0.0) |
|
240 µg/mL |
A |
2.40 |
150 |
1 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
1 |
0 |
B |
1.05 |
150 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
1 |
1 |
1 |
|
Total |
(32) |
300 |
1 |
1 |
0 |
0 |
0 |
0 |
2 |
1 |
2 (0.7) |
1 (0.3) |
|
Positive Control Cyclophosphamide (CP) 1.25 µg/mL |
A |
2.30 |
108a |
5 |
13 |
1 |
1 |
0 |
0 |
20 |
15 |
19 |
15 |
B |
1.65 |
99a |
12 |
18 |
0 |
1 |
0 |
0 |
31 |
19 |
23 |
16 |
|
Total |
(37) |
207 |
17 |
31 |
1 |
2 |
0 |
0 |
51 |
34 |
42 (20.3) |
31*** (15.0) |
|
aSlide evaluation terminated when 15 cells with aberrations (excluding gaps) had been observed
*** P < 0.001
DMSO Dimethyl sulphoxide
Table 7. Chromosome Aberration Test – Main Experiment 24-hour Continuous Exposure Without Metabolic Activation (S9) |
|||||||||||||
Treatment Group |
Replicate |
Mitotic Index (%) |
Number of cells scored |
Number of Aberrations |
Total Number of Aberrations |
Frequency of Aberrant Cells (%) |
|||||||
Gaps |
Chromatid |
Chromosome |
Others X |
(+Gaps) |
(-Gaps) |
(+Gaps) |
(- Gaps) |
||||||
Break |
Exchanges |
Breaks |
Exchanges |
||||||||||
Vehicle Control (DMSO) |
A |
5.60 |
150 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
1 |
1 |
1 |
B |
7.50 |
150 |
1 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
1 |
0 |
|
Total |
(100) |
300 |
1 |
1 |
0 |
0 |
0 |
0 |
2 |
1 |
2 (0.7) |
1 (0.3) |
|
15 µg/mL |
A |
4.00 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
4.70 |
150 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
1 |
1 |
1 |
|
Total |
(66) |
300 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
1 |
1 (0.3) |
1 (0.3) |
|
30 µg/mL |
A |
3.40 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
3.40 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Total |
(52) |
300 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 (0.0) |
0 (0.0) |
|
60 µg/mL |
A |
3.05 |
150 |
0 |
2 |
0 |
0 |
1 |
0 |
3 |
3 |
3 |
3 |
B |
3.00 |
150 |
3 |
0 |
0 |
1 |
0 |
0 |
4 |
1 |
4 |
1 |
|
Total |
(46) |
300 |
3 |
2 |
0 |
1 |
1 |
0 |
7 |
4 |
7 (2.3) |
4 (1.3) |
|
75 µg/mL |
A |
2.20 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
2.95 |
150 |
3 |
0 |
0 |
0 |
0 |
0 |
3 |
0 |
3 |
0 |
|
Total |
(39) |
300 |
3 |
0 |
0 |
0 |
0 |
0 |
3 |
0 |
3 (1.0) |
0 (0.0) |
|
Positive Control Mitomycin C (MMC) 0.1 |
A |
2.60 |
85a |
2 |
9 |
7 |
2 |
0 |
0 |
20 |
18 |
15 |
15 |
B |
2.40 |
63a |
2 |
11 |
5 |
1 |
0 |
0 |
19 |
17 |
17 |
15 |
|
Total |
(38) |
148 |
4 |
20 |
12 |
3 |
0 |
0 |
39 |
35 |
32 (21.6) |
30*** (20.3) |
|
aSlide evaluation terminated when 15 cells with aberrations (excluding gaps) had been observed
*** P < 0.001
DMSO Dimethyl sulphoxide
Table 8. Mean Frequency of Polyploid Cells (%) |
|||
Dose Level (µg/mL) |
Exposure Group |
||
4(20)-Hour Without S9 |
4(20)-Hour With S9 |
24-Hour Without S9 |
|
0 |
0 |
0 |
0 |
15 |
0 |
NA |
0 |
30 |
0 |
NA |
0 |
45 |
0 |
NA |
NA |
60 |
0 |
0 |
0 |
75 |
NA |
0 |
0 |
120 |
NA |
0 |
NA |
240 |
NA |
0 |
NA |
Mitomycin C (MMC) 0.4 |
0 |
NA |
NA |
Mitomycin C (MMC) 0.1 |
NA |
NA |
0 |
Cyclophosphamide (CP) 1.25 µg/mL |
NA |
0 |
NA |
NA Not applicable
Table 1. Preliminary Cytotoxicity Test Results |
|||
Dose (µg/mL) |
% RSG (-S9) 4-Hour Exposure |
% RSG (+S9) 4-Hour Exposure |
% RSG (-S9) 24-Hour Exposure |
0 |
100 |
100 |
100 |
7.81 |
69 |
111 |
73 |
15.63 |
54 |
103 |
32 |
31.25 |
29 |
108 |
5 |
62.5 |
8 |
88 |
0 |
125 |
2 |
22 |
0 |
250 |
2 |
3 |
0 |
500 |
1 |
1 |
0 |
1000 |
0 |
0 |
0 |
2000 |
0 |
0 |
0 |
Table 2. Main Experiment: Summary of Results |
|||||||||||
Treatment (µg/mL) |
4-hours (-S9) |
Treatment (µg/mL) |
4-hours (+S9) |
Treatment (µg/mL) |
24-hours (-S9) |
||||||
%RSG |
RTG |
MF§ |
%RSG |
RTG |
MF§ |
%RSG |
RTG |
MF§ |
|||
0 |
100 |
1.00 |
107.53 |
0 |
100 |
1.00 |
114.51 |
0 |
100 |
1.00 |
157.74 |
4 Ø |
88 |
|
|
4 Ø |
105 |
|
|
0.5 Ø |
103 |
|
|
8 Ø |
90 |
|
|
8 Ø |
103 |
|
|
1 |
105 |
0.95 |
161.34 |
16 |
71 |
0.62 |
142.01 |
16 |
109 |
1.21 |
104.40 |
2 |
99 |
0.93 |
174.58 |
32 |
43 |
0.39 |
135.55 |
32 |
94 |
0.92 |
120.90 |
4 |
76 |
0.82 |
162.76 |
40 |
42 |
0.38 |
142.98 |
40 |
96 |
0.86 |
155.57 |
8 |
62 |
0.68 |
154.32 |
48 |
36 |
0.32 |
138.73 |
48 |
92 |
0.82 |
127.98 |
16 |
20 |
0.30 |
195.23 |
56 |
22 |
0.17 |
180.16 * |
56 |
87 |
0.88 |
105.22 |
24 |
6 |
0.12 |
234.62 * |
64 X |
13 |
0.08 |
166.91 |
64 |
87 |
0.81 |
143.08 |
32 Ø |
4 |
|
|
Linear Trend *** |
Linear Trend NS |
Linear Trend ** |
|||||||||
EMS |
|
|
|
CP |
|
|
|
EMS |
|
|
|
400 |
74 |
0.45 |
831.33 |
1.5 |
85 |
0.60 |
958.32 |
150 |
47 |
0.41 |
1373.60 |
%RSG = Relative Suspension Growth
RTG = Relative Total Growth
MF§ = 5-TFT resistant mutants/106viable cells 2 days after exposure
X = Excluded due to excessive toxicity
Ø = Not plated for viability or 5-TFT resistance
CP = Cyclophosphamide
EMS = Ethylmethanesulphonate
* = P<0.05
** = P<0.01
*** = P<0.001
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
AMES Test
The test material Santicizer Platinum P1400 was evaluated for mutagenic activity in the vitro Salmonella-E coli/mammalian microsome reverse assay using the plate incorporation method (WIL Research, 2014a). In the initial assay, Santicizer Platinum P-1400 was tested at 25, 50, 100, 250, 500, 1000, 2500 and 5000 µg/plate using the plate incorporation method. Precipitates were observed at ≥500 µg/plate in all strains without metabolic activation and at ≥2500 µg/plate in all strains with metabolic activation. Cytotoxicity was observed at ≥1000 µg/plate in TA100 with and without metabolic activation.
In the confirmatory assay, Santicizer Platinum P-1400 was tested at 25, 50, 100, 250,500, 1000, 2500 and 5000 µg/plate using the plate incorporation method. Precipitates were observed at ≥500 µg/plate in all strains without metabolic activation and at ≥2500 µg/plate in all strains with metabolic activation. Cytotoxicity was observed at ≥250 µg/plate in TA1537 without metabolic activation, at ≥2500 µg/plate in TA100 with and without metabolic activation, and at 5000 µg/plate in TA1535 without metabolic activation.
In both assays, criteria for a negative response were met for all tester strains with and without metabolic activation. In conclusion, the test material Santicizer Platinum P1400 is considered to be negative for inducing mutagenicity under the condition of this study.
Chromosome Aberration
The study was conducted to detect the structural chromosomal aberrations in cultured mammalian cells (Envigo Research Limited, 2016j).
Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.
The dose levels used in the Main Experiment were selected using data from the preliminary cytotoxicity test where the results indicated that the maximum concentration should be based on cytotoxicity. All the positive control items induced statistically significant increases in the frequency of cells with chromosomal aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The results indicated that the test item was cytotoxic but did not induce any statistically significant increases in the frequency of cells with chromosomal aberrations, using a dose range that included a dose level that exceeded 55±5% mitotic inhibition. The vehicle (dimethyl sulphoxide) controls had cells with chromosomal aberration frequencies within the range expected for normal human lymphocytes, therefore the study is considered valid.
According to the conditions of this study, the test item, 1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2-(phenylmethyl) ester, CAS 1200806-67-2 was considered to be non-clastogenic to human lymphocytes in vitro.
Mouse Lymphoma Assay
The study was conducted to assess the mutagenicity potential of the test item 1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2-(phenylmethyl) ester, CAS 1200806-67-2 (Envigo Research Limited, 2016k). The test was done accoriding to the OECD Guidelines for Testing of Chemicals No. 490 "In Vitro Mammalian Cell Gene Mutation Tests using the Thymidine Kinase Gene, Method B17 of Commission Regulation (EC) No. 440/2008and the US EPA OPPTS 870.5300 Guideline. It is also aligned with the Japanise MITI/MHW Guideline.
In the main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (DMSO), and positive controls using 4 -hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 hour exposure group in the absence of metabolic activation. The dose range of the test item used in the main test was selected following the results of a preliminary toxicity study.
The maximum dose level used in the Mutagenicity Test was limited by test item induced toxicity in the absence of metabolic activation and by precipitate of the test item in the presence of metabolic activation. The mutant frequency values in the vehicle control groups were acceptable for the L5178Y cell line at the TK +/- locus. The positive controls induced marked increases in the mutant frequency, indicating the satisfactory performance of the test and of the activity of the metabolizing system. The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.
In conclusion, according to the results of this study, the test item 1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2-(phenylmethyl) ester, CAS 1200806-67-2 did not induce a toxicological statistically significant increase in the mutant frequency at the TK +/- locus in L5178Y cells.
Justification for classification or non-classification
P1400 does not meet the criteria for classification for mutagenicity under EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.
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