Registration Dossier
Registration Dossier
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 228-846-8 | CAS number: 6362-80-7
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: The test item was determined to be non-mutagenic under the conditions of this test.
in vitro chromosome aberration study: The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.
in vitro gene mutation study in mammalian cells: The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study performed according to GLP and guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- GLP compliance:
- yes
- Type of assay:
- bacterial gene mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Dose finding test:
Test concentrations in the dose-finding test were set in accordance with Methods for Testing New Chemical Substances (November 21, 2003) with 5000 μg/plate as the highest concentration for each strain and lower concentrations at 1250, 312.5, 78.1, 19.5, 4.88, 1.22 and 0.305 μg/plate in a common ratio of 4 for a total of 8 concentrations both with and without addition of S9 mix.
Main test (I) and Main Test (II):
From the results of the dose-finding test, 6 concentrations in a common ratio of 2 were set for the main tests (I) and (II) with at least 4 concentrations expected to show no growth inhibition of bacteria both with and without addition of S9 mix. Without S9 mix, concentrations were set at 9.77, 19.5, 39.1, 78.1, 156.3 and 312.5 μg/plate for TA100 and TA1535, at 156.3, 312.5, 625, 1250, 2500 and 5000 μg/plate for WP2uvrA, and at 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 μg/plate for TA98 and TA1537. With S9 mix, they were set at 39.1, 78.1, 156.3, 312.5, 625 and 1250 μg/plate for TA100, at 156.3, 312.5, 625, 1250, 2500 and 5000 μg/plate for TA1535 and WP2uvrA, at 9.77, 19.5, 39.1, 78.1, 156.3 and 312.5 μg/plate for TA98, and at 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 μg/plate for TA1537. Negative controls and positive controls were provided for each strain. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- other: 9-aminoacridine hydrochloride and 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide
- Untreated negative controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Details on test system and experimental conditions:
- The test was conducted by the preincubation method without metabolic activation (without S9 mix) and with metabolic activation (with S9 mix). (1) 0.1 mL of sample solution, (2) 0.5 mL of high-pressure steam sterilized 0.1 mol/L sodium phosphate buffer solution (pH 7.4) (without metabolic activation) or 0.5 mL of S9 mix (with metabolic activation) and (3) 0.1 mL of bacterial suspension were added in that order to dry-heat sterilized test tubes (15.5100 mm, clean test tube Rarubo, Terumo Corp.), which were incubated at 37°C for 20 minutes using a shaker in reciprocating mode. Then 2 mL of top agar warmed to 45°C was added and mixed, spread on the minimum glucose agar plate medium, and the plate was overturned and incubated for about 48 hours in an incubator set at 37°C.
After the end of incubation, the presence or absence of precipitation on the plate was observed visually, then the number of revertant colonies was counted with a colony analyzer (CA-11D, System Science Co., Ltd.), and the bacteria were observed for growth inhibition under a 100x microscope. The plates were also visually observed for precipitation at the start of incubation.
The number of plates for each strain at each concentration with and without metabolic activation was one in the dose-finding test and three in both main tests (I) and (II). Test tubes and plates were identified by colors marked in oil-based ink for each strain. - Evaluation criteria:
- Test results were considered positive if the number of revertant colonies in the plates treated with test substance was twice or more that of the negative control, and dose-dependent increase was observed
- Statistics:
- Mean and standard deviation of the numbers of revertant colonies were calculated for each concentration. Significance of difference was not tested because the following judgment criteria were used
- Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Dose-Finding Test (Table 1)
Precipitation on the Plate
Without S9 mix, a clear oily precipitate was observed on the plates at concentrations of 1250 μg/plate and above both at the start and end of incubation. With S9 mix, a white oily film of precipitate was observed at 1250 μg/plate and above at the start of incubation, and a fine white precipitate was observed at the highest concentration of 5000 μg/plate at the end of incubation.
Bacterial Growth Inhibition
Bacterial growth inhibition was observed without S9 mix at 312.5 μg/plate and above for TA100 and TA1535, at the highest concentration of 5000 μg/plate for WP2uvrA, and at 78.1 μg/plate and above for TA98 and TA1537. With S9 mix, it was observed at 1250 μg/plate and above for TA100, at the highest concentration of 5000 μg/plate for TA1535 and WP2uvrA, at 312.5 μg/plate and above for TA98, and at 78.1 μg/plate and above for TA1537.
Number of Revertant Colonies
The number of revertant colonies was less than twice that of the negative control for any strains with or without S9 mix.
Sterility Test
No bacterial contamination of the highest concentration of test substance solution (50 μg/mL) or the S9 mix was observed in the sterility test.
Negative Control and Positive Controls
The positive control substances showed clear increases in the numbers of revertant colonies, and the numbers of revertant colonies in the negative control and positive controls were within the range of the background data of the test facility.
Main Test (I) (Tables 2-1 to 2-2 and Main Test (II) (Tables 3-1 to 3-2 )
Precipitation on the Plate
Without S9 mix, a clear oily precipitate was observed on the plates at concentrations of 625 μg/plate and above at the start of incubation and at 1250 μg/plate and above at the end of incubation. With S9 mix, a white oily film of precipitate was observed at 1250 μg/plate and above at the start of incubation, and a fine white precipitate was observed at 2500 μg/plate and above at the end of incubation.
Bacterial Growth Inhibition
Bacterial growth inhibition was observed without S9 mix at 156.3 μg/plate and above for TA100 and TA1535, at the highest concentration of 5000 μg/plate for WP2uvrA, and at 39.1 μg/plate and above for TA98 and TA1537. With S9 mix, it was observed at 625 μg/plate and above for TA100, at 2500 μg/plate and above for TA1535, at the highest concentration of 5000 μg/plate for WP2uvrA, at the highest concentration of 312.5 μg/plate for TA98, and at the highest concentration of 78.1 μg/plate for TA1537.
Number of Revertant Colonies
The number of revertant colonies was less than twice that of the negative control for all strains with or without S9 mix.
Sterility Test
No bacterial contamination of the highest concentration of test substance solution (50 μg/mL) or the S9 mix was observed in the sterility test.
Negative Control and Positive Controls
The positive control substances showed clear increases in the numbers of revertant colonies, and the numbers of revertant colonies in the negative control and positive controls were within the range of the background data of the test facility. - Conclusions:
- From these results, 2,4-diphenyl-4-methyl-1-pentene is judged to have no potential to induce genetic mutations under the conditions of this study
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study performed according to GLP and guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Based on the 50% cell growth inhibition concentrations and survival rate from the results of the cell growth inhibition test, five test concentrations were set in a common ratio of 2 at 12.5, 25, 50, 100 and 200 μg/mL for short treatment with S9 mix, 6.25, 12.5, 25, 50 and 100 μg/mL for short treatment without S9 mix, and 3.13, 6.25, 12.5, 25 and 50 μg/mL for continuous treatment (24-hour treatment)
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- yes
- Remarks:
- Dimethyl sulfoxide
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- N-dimethylnitrosamine
- mitomycin C
- Details on test system and experimental conditions:
- The test was conducted using 3 systems: short treatment with and without S9 mix and continuous treatment for 24 hours
Short Treatment Method:
After removing 2.5 mL of culture medium from the plates with S9 mix, 0.5 mL of S9 mix and 15 μL for test substance and negative control substance, or 0.3 mL for positive control substance, were added. 2.0 mL of culture medium was removed from plates without S9 mix, and test solution was added in the same manner as with S9 mix. All plates were incubated in the CO2 incubator for 6 hours, then 5.0 mL of culture medium was replaced with fresh medium and plates were incubated for a further 18 hours before preparing chromosome specimens and determining of the number of viable cells. Plates were observed visually for the presence of precipitation of test substance at the time of adding test solutions and at the end of treatment.
Continuous Treatment Method:
25 μL for test substance and negative control substance, or 0.5 mL for positive control substance, were added to the plates, which were incubated in the CO2 incubator for 24 hours before preparing chromosome specimens and determining of the number of viable cells. Plates were observed visually for the presence of precipitation of test substance at the time of adding test solutions and at the end of treatment.
Preparation of Specimens:
In both the short treatment and continuous treatment method, 0.1 mL of 10 μg/mL Colcemid solution was added to each plate 2 hours before the end of incubation to obtain metaphase cells. After the end of incubation, approximately 3 mL of 0.25% trypsin solution was added to each plate to harvest the cells, which were recovered by centrifugation and subjected to hypotonic treatment with 75 mmol/L aqueous potassium chloride solution (37°C, 15 min). After the end of hypotonic treatment, the cells were centrifuged again, mixed with methanol and acetic acid in a ratio of 3:1, dehydrated and fixed with ice-cooled fixative, and cell suspensions were prepared with the same fixative. One drop of this cell suspension was placed on each of two places on a glass slide, and the cells were spread, dried, and stained with 2% Giemsa stain for approximately 15 minutes.
Three slides were prepared for each plate and assigned random code numbers. After the end of observation, labels indicating the study number, name of test substance or control substance, concentration, test method, presence of absence of S9 mix (short treatment) or incubation time (continuous treatment), preparation date of specimens were affixed to the slides.
Determination of Cell Count:
In both short treatment and continuous treatment, approximately 3 mL of 0.25% trypsin solution was added to each plate after the end of incubation to harvest the cells. Cells were recovered by centrifugation and viable cell counts were determined using a hemocytometer.
Measurement was conducted 3 times for each plate, and the cell survival rate at each concentration was obtained using the mean value, with the viable cell count in the negative control group taken as 100%.
Observation of Specimens:
100 cells with chromosomes in well-spread metaphase were observed for each plate, 200 cells for each concentration. Specimens were observed in the order of code number, and collation of the results of observation and test concentrations was done after the end of observation for each treatment method.
Chromosomal aberrations were classified as numerical aberrations and structural aberrations. Numerical aberrations observed were polyploidy and endoreduplication. Structural aberrations were classified as (1) gaps (gap), chromatid breaks (ctb), (3) chromosome breaks (csb), (4) chromatid exchange (cte), (5) chromosome exchange (cse) and (6) fragmentation (frg). A cell with any of these chromosomal aberrations was recorded as one positive cell. In distinguishing gaps and breaks, a gap was a segment that is aligned with the axis of the chromosome or chromatid and a break was a misaligned segment, but an achromatic segment wider than the width of the chromatid was considered a break even if it was not misaligned. The total number of chromosomal aberrations was recorded with and without the inclusion of gaps
Test Validity Conditions:
The test was considered valid if the incidence of cells with chromosomal aberrations in the negative control group was less than 5%, the incidence of cells with chromosomal aberrations excluding gaps in the positive control group was at least 10%, the incidence of chromosomal aberrations in the negative and positive control groups is within the range of the background data of the test facility (Attachment 5), and there were no other factors affecting the test systems. - Evaluation criteria:
- The results of the test were considered negative if the incidence of cells with numerical or structural aberrations was less than 5% and false-positive if the incidence was 5% or more but less than 10%. Incidence of 10% or more which showed dose-dependent increase was regarded as positive. The incidence of cells with structural aberrations was calculated both including and excluding gaps, but evaluation was done using the incidence excluding gaps
- Statistics:
- Significance of difference in the incidence of cell having chromosomal aberrations was not tested because the following judgment criteria were used
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Short term treatment method (test results are shown in Table 3 (see attached background information):
The incidence of cells with numerical aberrations in the 2,4-diphenyl-4-methyl-1-pentene treatment groups was negative at 1.0% or less at all concentrations with or without S9 mix. Similarly, the incidence of cells with structural aberrations was negative at 1.5% or less at all concentrations with or without S9 mix.
The survival rate of cells in the 2,4-diphenyl-4-methyl-1-pentene treatment groups with S9 mix was 90% or more at 12.5 and 25 μg/mL, but decreased with the increase in concentration at 50 μg/mL and higher, and was 30% at the highest concentration of 200 μg/mL. The survival rate of cells with S9 mix was 90% or more at 6.25 and 12.5 μg/mL, but decreased with the increase in concentration at 25 μg/mL and higher, and was 31% at the highest concentration of 100 μg/mL.
Precipitation of the test substance was not observed at any concentration with or without S9 mix at the time of test solution addition or at the end of treatment.
The incidence of cells with numerical aberrations in the negative control group was 1.0% with S9 mix and 0.5% without S9 mix. The incidence of cells with structural aberrations was 0.5% with S9 mix and 0% without S9 mix. The incidence of cells with numerical aberrations in the positive control groups (with S9 mix: DMN 500 μg/mL, without S9 mix: MMC 0.1 μg/mL) was 0.5% with S9 mix and 0% without S9 mix. The incidence of cells with structural aberrations was 68.5% with S9 mix and 52.5% without S9 mix.
The induction of chromosomal aberrations in the negative and positive controls was mostly within the background data, and the test conditions were fulfilled.
Continuous Treatment Method (test results are showb in Table 4 (see attached background information)
The incidence of cells with numerical aberrations in the 2,4-diphenyl-4-methyl-1-pentene treatment groups was negative at 0.5% or less at all concentrations. Similarly, the incidence of cells with structural aberrations was negative at 1.0% or less at all concentrations.
The survival rate of cells in the 2,4-diphenyl-4-methyl-1-pentene treatment groups was 90% or more at 3.13 and 6.25 μg/mL, but decreased with the increase in concentration at 12.5 μg/mL and higher, and was 27% at the highest concentration of 50 μg/mL.
Precipitation of the test substance was not observed at any concentration at the time of test solution addition or at the end of treatment.
The incidence of cells with numerical aberrations in the negative control group was 0% and the incidence of cells with structural aberrations was also 0%. The incidence of cells with numerical aberrations in the positive control group (MMC 0.05 μg/mL) was 0% and the incidence of cells with structural aberrations was 46.5%.
The incidence of chromosomal aberrations in the negative and positive controls was mostly within the background data, and the test conditions were fulfilled.
- Conclusions:
- The incidence of cells with numerical aberrations and structural aberrations in the 2,4-diphenyl-4-methyl-1-pentene treatment groups was less than 5% in each test system, and therefore 2,4-diphenyl-4-methyl-1-pentene is considered not to induce either numerical or structural aberrations.
The incidence of cells with aberrations in the negative and positive control groups was mostly within the background data in each test system in both the continuous treatment and short treatments, and the conditions of test validity were fulfilled.
From the results, it is judged that 2,4-diphenyl-4-methyl-1-pentene does not induce chromosomal aberrations under the conditions of this study. - Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- The experimental phases of the study were performed between 10 September 2012 and 21 February 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: 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
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) are used during the course of the study.
- Test concentrations with justification for top dose:
- - Preliminary toxicity test: 9.22 to 2360 µg/mL
- Experiment 1, without metabolic activation: 2.5, 5, 7.5, 10, 12.5, 15, 17.5 and 20 µg/mL
- Experiment 1, with metabolic activation: 2.5, 5, 10, 15, 20, 25, 30 and 35 µg/mL
- Experiment 2, without metabolic activation: 2.5, 5, 7.5, 10, 15, 17.5, 20 and 25 µg/mL
- Experiment 2, with metabolic activation: 5, 10, 15, 20, 25, 30, 35 and 40 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- Sigma batch BCBH3157V at 400 μg/mL and 150 μg/mL for Experiment 1 and Experiment 2, respectively, was used as the positive control in the absence of metabolic activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- Acros batch A0302605 at 2 μg/mL was used as the positive control in the presence of metabolic activation for Experiments 1 and 2
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
Preliminary toxicity test
- Exposure duration: 4 h with and without metabolic activation (S9); 24 h without S9
- Expression time (cells in growth medium): 24 h
Experiment 1
- Exposure duration: 4 h
- Expression time (cells in growth medium): 2 d
- Selection time (if incubation with a selection agent): 10 to 14 d
Experiment 2
- Exposure duration: 4 h with metabolic activation; 24 h without metabolic activation
- Expression time (cells in growth medium): 2 d
- Selection time (if incubation with a selection agent): 10 to 14 d
SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)
STAIN (for cytogenetic assays): 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS)
NUMBER OF REPLICATIONS: The treatments were performed in duplicate (A + B), both with and without metabolic activation at 8 dose levels of the test item, vehicle and positive controls
NUMBER OF CELLS EVALUATED: on day 2 of the experiment, the cells were counted, diluted to 10^4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL TFT in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (% V) in non-selective medium
DETERMINATION OF CYTOTOXICITY
- Method: The daily cell counts were used to obtain a Relative Suspension Growth (% RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (% V) data a Relative Total Growth (RTG) value.
OTHER
Preliminary toxicity test
- A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/mL, using a 4 h exposure period both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/mL using a 24 h exposure period without S9. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted using a Coulter counter and then serially diluted to 2 x 10^5 cells/mL.
- The cultures were incubated at 37 °C with 5 % CO2 in air and sub-cultured after 24 h by counting and diluting to 2 x 10^5 cells/mL. After a further 24 h 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 treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (% RSG) value.
- Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) Maximum recommended dose level, 5000 μg/mL or 10 mM.
ii) The presence of excessive precipitate where no test item-induced toxicity was observed.
iii) Test item-induced toxicity, where the maximum dose level used should produce 10 to 20 % survival (the maximum level of toxicity required). This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al 2002)
Experiment 1
- Several 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 universals. The treatments were performed in duplicate (A + B), both with and without metabolic activation (2 % S9 final concentration) at 8 dose levels of the test item (2.5 to 20 μg/mL in the absence of metabolic activation, and 2.5 to 35 μg/mL in the presence of metabolic activation), vehicle and positive controls. To each universal was added 2 mL of S9-mix if required, 0.2 mL of the treatment dilutions, (0.2 mL for the positive control) and sufficient R0 medium to bring the total volume to 20 mL.
- The treatment vessels were incubated at 37 °C for 4 h with continuous shaking using an orbital shaker within an incubated hood.
- At the end of the treatment period 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 h for the expression period of 2 d, by counting and dilution 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.
Experiment 2
- An exponentially growing stock culture of cells was established. The cells were counted and processed to give 1 x 10^6 cells/mL in 10 mL cultures in R10 medium for the 4 h treatment with metabolic activation cultures. In the absence of metabolic activation the exposure period was extended to 24 h therefore 0.3 x 10^6 cells/mL in 10 mL cultures were established in 25 cm^2 tissue culture flasks. The treatments were performed in duplicate (A + B), both with and without metabolic activation (2 % S9 final concentration) at 8 dose levels of the test item (2.5 to 25 μg/mL in the absence of metabolic activation and 5 to 40 μg/mL in the presence of metabolic activation), vehicle and positive controls. To each culture vessel was added 2 mL of S9-mix if required, 0.2 mL of the treatment dilutions, (0.2 mL for the positive control) and sufficient R0 medium to give a final volume of 20 mL (R10 is used for the 24 h exposure group).
- The treatment vessels were incubated at 37 °C with continuous shaking using an orbital shaker within an incubated hood for 24 h in the absence of metabolic activation and 4 h in the presence of metabolic activation
- At the end of the treatment period 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 h for the expression period of 2 d, by counting and dilution 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. - Evaluation criteria:
- For a test item to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. The IMF must exceed some value based on the global background MF for each method (agar or microwell). The Global Evaluation Factor (GEF) value is 126 x 10^-6 for the microwell method.
Any test item dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10^-6 and demonstrate a positive linear trend will be considered positive.
If a test item produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance.
When a test item induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant. - 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:
- PRELIMINARY TOXICITY TEST: In all 3 of the exposure groups there was evidence of marked dose-related reductions in the Relative Suspension Growth (% RSG) of cells treated with the test item when compared to the concurrent vehicle controls. The steep nature of the toxicity curve was taken to indicate that achieving optimum toxicity would be difficult. Overall, precipitate of the test item was observed at and above 147.5 μg/mL. Based on the % RSG values observed, the maximum dose levels in the subsequent Mutagenicity Test were limited by test item induced toxicity.
EXPERIMENT 1: There was evidence of marked dose-related toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the RTG and % RSG values. There was evidence of a modest reduction in viability (% V) in the presence of metabolic activation indicating that residual toxicity had occurred in this exposure group. Based on the % RSG and RTG values observed, optimum levels of toxicity were considered to have been achieved in both the absence and presence of metabolic activation. Acceptable levels of toxicity were seen with both positive control substances.
The vehicle (solvent) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.
The test item did not induce any statistically significant dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell in either the absence or presence of metabolic. A statistically significant increase in mutant frequency was observed at an individual dose level, the upper dose level, in the absence of metabolic activation. However, the GEF was not exceeded, there was no evidence of any significant increases in absolute numbers of mutant colonies and the mutant frequency value observed would have been considered acceptable for vehicle controls. The response was considered to be artefactual and of no toxicological significance. Precipitate of the test item was not observed at any of the dose levels.
EXPERIMENT 2: There was evidence of marked toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the % RSG and RTG values. There was evidence of modest reductions in viability (%V) in the presence of metabolic activation indicating that residual toxicity had occurred in this exposure group. Based on the % RSG and RTG values observed, optimum levels of toxicity were considered to have been achieved in both the absence and presence of metabolic activation. The excessive toxicity observed at and above 20 μg/mL in the absence of metabolic activation, and at 40 μg/mL in the presence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance. The RTG value observed at 35 μg/mL in the presence of metabolic activation exceeded the upper acceptable limit of acceptable toxicity and was therefore excluded from the statistical analysis. Acceptable levels of toxicity were seen with both positive control substances.
The 24-hour exposure without metabolic activation demonstrated that the extended time point had a modest effect on the toxicity of the test item.
The vehicle (solvent) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.
The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell at any of the dose levels in the absence of metabolic activation. A very modest but statistically significant dose related (linear-trend) increase in mutant frequency was observed in the presence of metabolic activation. However, statistically significant increases in mutant frequency were not observed at any of the individual dose levels and the GEF was also not exceeded at any of the dose levels, including the dose level that achieved optimum levels of toxicity. There was also no evidence of any significant increases in absolute numbers of mutant colonies and the mutant frequency values observed at dose levels with acceptable levels of toxicity would have been considered acceptable for vehicle controls. The response was, therefore, considered to be artefactual and of no toxicological significance. Precipitate of the test item was not observed at any of the dose levels. - Conclusions:
- The potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line was assessed according to OECD guideline 476. The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.
Referenceopen allclose all
Table 1: Reverse mutation test of 1,1’-(1,1-dimethyl-3-methylene-1,3-propanediyl) bisbenzene in bacteria (dose range finding test)
With (+) or without (-) S9 mix |
Compound concentration (µg/plate) |
Number of revertants (number of colonies/plate) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||
S9 mix (-) |
Negative control |
111 |
10 |
51 |
19 |
13 |
0.305 |
136 |
17 |
48 |
17 |
3 |
|
1.22 |
146 |
8 |
46 |
9 |
6 |
|
4.88 |
125 |
16 |
35 |
17 |
9 |
|
19.5 |
118 |
13 |
47 |
20 |
13 |
|
78.1 |
137 |
17 |
44 |
13* |
14* |
|
312.5 |
114* |
10* |
33 |
22* |
8* |
|
1250# |
126* |
13* |
49 |
16* |
7* |
|
5000# |
125* |
16* |
48* |
22* |
6* |
|
S9 mix (+) |
Negative control |
146 |
9 |
41 |
29 |
20 |
0.305 |
137 |
16 |
36 |
26 |
15 |
|
1.22 |
138 |
16 |
44 |
31 |
18 |
|
4.88 |
154 |
14 |
49 |
32 |
19 |
|
19.5 |
127 |
11 |
46 |
29 |
21 |
|
78.1 |
137 |
17 |
40 |
28 |
19* |
|
312.5 |
139 |
7 |
42 |
17* |
20* |
|
1250# |
123* |
12 |
44 |
35* |
10* |
|
5000# |
137* |
12* |
36* |
27* |
4* |
|
Positive control not requiring S9 mix |
Name |
AF-2 |
NaN3 |
AF-2 |
AF-2 |
9AA |
Concentration (µg/plate) |
0.01 |
0.5 |
0.01 |
0.1 |
80 |
|
Number of colonies/plate |
531 |
628 |
157 |
485 |
571 |
|
Positive control requiring S9 mix |
Name |
2AA |
||||
Concentration (µg/plate) |
1 |
2 |
10 |
0.5 |
2 |
|
Number of colonies/plate |
957 |
312 |
843 |
352 |
187 |
|
Negative control: Dimethylsulfoxide
AF2:2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide; NaN3sodium azide; 9AA: 9-aminoacridine hydrochloride; 2AA: 2-aminoanthracene.
*: Bacterial growth inhibition was observed
#: Clear oily precipitations were observed on the surface of agar plate
##: White fine precipitations were observed on the surface of agar plate
Table 2-1: Reverse mutation test of 1,1’-(1,1-dimethyl-3-methylene-1,3-propanediyl) bisbenzene in bacteria (mutagenicity test I: -S9 mix)
Compound concentration (µg/plate) |
Number of revertants (number of colonies/plate) |
|||||
Base-pair substitution type |
Frameshift type |
|||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||
Negative control |
117, 118, 143 (126 ± 14.7) |
12, 17, 19 (16 ± 3.6) |
43, 45, 46 (45 ± 1.5) |
13, 19, 22 (18 ± 4.6) |
13, 14, 20 (16 ± 3.8) |
|
2.44 |
|
|
|
16, 21, 27 (21 ± 5.5) |
17, 19, 24 (20 ± 4.6) |
|
4.88 |
|
|
|
20, 25, 28 (24 ± 4.0) |
19, 25, 28 (24 ± 4.6) |
|
9.77 |
97, 104, 111 (104 ± 7.0) |
15, 18, 24 (19 ± 4.6) |
|
16, 20, 20 (19 ± 2.3) |
16, 20, 23 (20 ± 5.9) |
|
19.5 |
114, 116, 130 (120 ± 8.7) |
20, 20, 25 (22 ± 2.9) |
|
17, 19, 19 (18 ± 1.2) |
18, 20, 29 (22 ± 5.9) |
|
39.1 |
100, 100, 103 (101 ± 1.7) |
13, 13, 17 (14 ± 2.3) |
|
20*, 21*, 21* (21 ± 0.6) |
21*, 22*, 23* (22 ± 5.9) |
|
78.1 |
97, 105, 118 (107 ± 10.6) |
12, 14, 18 (15 ± 3.1) |
|
15*, 19*, 27* (20 ± 6.1) |
15*, 17*, 24* (19 ± 4.7) |
|
156.3 |
88*, 101*, 104* (98± 8.5) |
11*, 16*, 19* (15 ± 4.0) |
44, 50, 60 (51 ± 8.1) |
|
|
|
312.5 |
90*, 109*, 109* (103 ± 11.0)
|
15*, 16*, 17* (16 ± 1.0) |
39, 47, 51 (46 ± 6.1) |
|
|
|
625 |
|
|
37, 51, 54 (47 ± 9.1) |
|
|
|
1250# |
|
|
54, 58, 67 (60 ± 6.7) |
|
|
|
2500# |
|
|
38, 41, 44 (41 ± 3.0) |
|
|
|
5000# |
|
|
46*, 55, 61* (54 ± 7.5) |
|
|
|
Positive control |
||||||
Name |
AF2 |
NaN3 |
AF-2 |
AF-2 |
9AA |
|
Concentration (µg/plate) |
0.01 |
0.5 |
0.01 |
0.1 |
80 |
|
Number of colonies/plate |
470, 544, 548 (521 ± 43.9) |
578, 604, 614 (599 ± 18.6) |
165, 168, 171 (168 ± 3.0) |
401, 425, 441 (422 ± 20.1) |
339, 393, 481 (404 ± 71.7) |
|
Negative control: Dimethylsulfoxide
AF2:2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide; NaN3sodium azide; 9AA: 9-aminoacridine hydrochloride.
( ): Mean ± S.D
*: Bacterial growth inhibition was observed
#: Clear oily precipitations were observed on the surface of agar plate
Table 2-2: Reverse mutation test of 1,1’-(1,1-dimethyl-3-methylene-1,3-propanediyl) bisbenzene in bacteria (mutagenicity test I: ±S9 mix)
Compound concentration (µg/plate) |
Number of revertants (number of colonies/plate) |
|||||
Base-pair substitution type |
Frameshift type |
|||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||
Negative control |
123, 130, 145 (133 ± 11.2) |
16, 17, 19 (17 ± 1.5) |
34, 42, 60 (45 ± 13.3) |
29, 36, 41 (35 ± 6.0) |
20, 21, 21 (21 ± 0.6) |
|
2.44 |
|
|
|
|
21, 33, 36 (30 ± 7.9) |
|
4.88 |
|
|
|
|
28, 33, 38 (33 ± 5.0) |
|
9.77 |
|
|
|
25, 37, 44 (35 ± 9.6) |
24, 29, 32 (28 ± 4.0) |
|
19.5 |
|
|
|
22, 26, 27 (25 ± 2.6) |
24, 29, 32 (28 ± 4.2) |
|
39.1 |
108, 126, 131 (122 ± 12.1) |
|
|
36, 36, 42 (38 ± 3.5) |
25, 26, 29 (27 ± 2.1) |
|
78.1 |
122, 123, 128 (124 ± 3.2) |
|
|
35, 36, 41 (37 ± 3.2) |
30*, 32*, 33* (32 ± 1.5) |
|
156.3 |
118, 124, 124 (122 ± 3.5) |
13, 18, 20 (17 ± 3.6) |
43, 51, 53 (49 ± 5.3) |
29, 30, 41 (33 ± 6.7) |
|
|
312.5 |
101, 111, 114 (109 ± 6.8) |
9, 10, 14 (11 ± 2.6) |
41, 46, 51 (46 ± 5.0) |
19*, 26*, 35* (27 ± 8.0) |
|
|
625 |
98*, 101*, 114* (104 ± 8.5) |
18, 18, 21 (19 ± 1.7) |
28, 37, 58 (41 ± 15.4) |
|
|
|
1250# |
82*, 86*, 97* (88 ± 7.8) |
7, 13, 18 (13 ± 5.5) |
42, 45, 54 (47 ± 6.2) |
|
|
|
2500# |
|
14*, 22*, 27* (21 ± 6.6) |
51, 56, 57 (55 ± 3.2) |
|
|
|
5000## |
|
15*, 17*, 19* (17 ± 2.0) |
42*, 53*, 69* (55 ± 13.6) |
|
|
|
Positive control |
||||||
Name |
2AA |
|||||
Concentration (µg/plate) |
1 |
2 |
10 |
0.5 |
2 |
|
Number of colonies/plate |
748, 777, 835 (787 ± 44.3) |
303, 330, 367 (333 ± 32.1) |
746, 759, 924 (810 ± 99.2) |
364, 381, 385 (377 ± 11.2) |
134, 142, 165 (147 ± 16.1) |
|
Negative control: Dimethylsulfoxide
2AA: 2-Aminoanthracene
( ): Mean ± S.D
*: Bacterial growth inhibition was observed
##: White fine precipitations were observed on the surface of agar plate
Table 3-1: Table 2-2: Reverse mutation test of 1,1’-(1,1-dimethyl-3-methylene-1,3-propanediyl) bisbenzene in bacteria (mutagenicity test II: -S9 mix)
Compound concentration (µg/plate) |
Number of revertants (number of colonies/plate) |
||||
Base-pair substitution type |
Frameshift type |
||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|
Negative control |
119, 119, 124 (121 ± 2.9) |
11, 12, 13 (12 ± 1.0) |
42, 49, 54 (48 ± 6.0) |
20, 21, 21 (21 ± 0.6) |
10, 16, 19 (15 ± 4.6) |
2.44 |
|
|
|
12, 18, 19 (16 ± 3.8) |
20, 22, 25 (22 ± 2.5) |
4.88 |
|
|
|
17, 23, 30 (23 ± 6.5) |
25, 26, 28 (26 ± 1.5) |
9.77 |
106, 114, 120 (113 ± 7.0) |
14, 16, 19 (16 ± 2.5) |
|
19, 21, 24 (21 ± 2.5) |
18, 20, 24 (21 ± 3.1) |
19.5 |
126, 127, 130 (128 ± 2.1) |
13, 13, 16 (14 ± 1.7) |
|
16, 22, 25 (21 ± 4.6) |
17, 21, 21 (20 ± 2.3) |
39.1 |
118, 136, 137 (130 ± 10.7) |
12, 14, 19 (15 ± 3.6) |
|
20*, 22*, 25* (22 ± 2.5) |
20*, 21*. 27* (22 ± 3.8) |
78.1 |
118, 120, 122 (120 ± 2.0) |
10, 14, 17 (14 ± 3.5) |
|
18*, 21*, 25* (21 ± 3.5) |
20*, 23*, 27* (23 ± 3.5) |
156.3 |
119*, 128*, 134* (127 ± 7.5) |
11*, 12*, 15* (13 ± 2.1) |
50, 61, 63 (58 ± 7.0) |
|
|
312.5 |
97*, 109*, 118* (108 ± 10.5) |
9*, 11*, 12* (11 ± 1.5) |
47, 55, 57 (53 ± 5.3) |
|
|
625 |
|
|
51, 63, 63 (59 ± 6.9) |
|
|
1250# |
|
|
53, 64, 72 (63 ± 9.5) |
|
|
2500# |
|
|
62, 69, 76 (69 ± 7.0) |
|
|
5000# |
|
|
56*, 58*, 62* (59 ± 3.1) |
|
|
Positive control |
|||||
Name |
AF-2 |
NaN3 |
AF-2 |
AF-2 |
9AA |
Concentration (µg/plate) |
0.01 |
0.5 |
0.01 |
0.1 |
80 |
Number of colonies/plate |
539, 549, 595 (561 ± 29.9) |
588, 604, 621 (604 ± 16.5) |
153, 169, 174 (165 ± 11.0) |
377, 387, 459 (408 ± 44.7) |
377, 435, 482 (431 ± 52.6) |
Negative control: Dimethylsulfoxide
AF2:2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide; NaN3sodium azide; 9AA: 9-aminoacridine hydrochloride.
( ): Mean ± S.D
*: Bacterial growth inhibition was observed
#: Clear oily precipitations were observed on the surface of agar plate
Table 3-2: Reverse mutation test of 1,1’-(1,1-dimethyl-3-methylene-1,3-propanediyl) bisbenzene in bacteria (mutagenicity test II: +S9 mix)
Compound concentration (µg/plate) |
Number of revertants (number of colonies/plate) |
||||
Base-pair substitution type |
Frameshift type |
||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|
Negative control |
107, 116, 140 (121 ± 17.1) |
10, 12, 13 (12 ± 1.5) |
48, 50, 52 (50 ± 2.0) |
30, 37, 38 (35 ± 4.4) |
20, 20, 27 (22 ± 4.0) |
2.44 |
|
|
|
|
26, 31, 32 (30 ± 3.2) |
4.88 |
|
|
|
|
23, 33, 35 (30 ± 6.4) |
9.77 |
|
|
|
29, 31, 32 (31 ± 1.5) |
25, 30, 37 (31 ± 6.0) |
19.5 |
|
|
|
28, 32, 37 (32 ± 4.5) |
32, 34, 41 (36 ± 4.7) |
39.1 |
144, 155, 164 (154 ± 10.0) |
|
|
33, 35, 42 (37 ± 4.7) |
25, 28, 36 (30 ± 5.7) |
78.1 |
128, 151, 159 (146 ± 16.1) |
|
|
35, 39, 39 (38 ± 2.3) |
31*, 34*, 37* (34 ± 3.0) |
156.3 |
115, 136, 141 (131 ± 13.8) |
8, 12, 19 (13 ± 5.6) |
47, 53, 61 (54 ± 7.0) |
29, 38, 40 (36 ± 5.9) |
|
312.5 |
123, 131, 142 (132 ± 9.5) |
8, 10, 11 (10 ± 1.5) |
60, 64, 67 (64 ± 3.5) |
25*, 38*, 38* (34 ± 7.5) |
|
625 |
109*, 137*, 151* (132 ± 21.4) |
9, 10, 12 (10 ± 1.5) |
50, 56, 60 (55 ± 5.0) |
|
|
1250# |
116*, 124*, 137* (126 ± 10.6) |
7, 9, 16 (11 ± 4.7) |
45, 51, 61 (52 ± 8.1) |
|
|
2500## |
|
10*, 11*, 11* (11 ± 0.6) |
34, 46, 47 (42 ± 7.2) |
|
|
5000## |
|
7*, 10*, 12* (10 ± 2.5) |
48*, 56*, 59* (54 ± 5.7) |
|
|
Positive control |
|||||
Name |
2AA |
||||
Concentration (µg/plate) |
1 |
2 |
10 |
0.5 |
2 |
Number of colonies/plate |
818, 903, 914 (878 ± 52.5) |
297, 317, 338 (317 ± 20.5) |
822, 832, 910 (855 ± 48.2) |
375, 376, 399 (388 ± 13.6) |
147, 153, 162 (154 ± 7.5) |
Negative control: Dimethylsulfoxide
2AA: 2-Aminoanthracene
( ): Mean ± S.D
*: Bacterial growth inhibition was observed
##: White fine precipitations were observed on the surface of agar plate
Table 1: Cell growth inhibition test of 1,1’- (1,1-dimethyl-3-methylene-1,3-propanediyl) bisbenzene in cultured CHL cells –The short treatment method-
Test substance |
Concentration (µg/mL) |
Treated for 6 hr with S9 mix |
Treated for 6 hr without S9 mix |
||||
No of cells (x104/plate) |
Survival ratioa)(%) |
IC50 (µg/mL) |
No of cells (x104/plate) |
Survival ratioa)(%) |
IC50 (µg/mL) |
||
Negative control (Dimethyl sulfoxide) |
- |
68 |
100 |
- |
64 |
100 |
- |
1,1’- (1,1-dimethyl-3-methylene-1,3-propanediyl) bisbenzene |
4.69 |
68 |
100 |
101.5 |
62 |
97 |
52.6 |
9.38 |
65 |
96 |
62 |
97 |
|||
18.8 |
64 |
94 |
61 |
95 |
|||
37.5 |
60 |
88 |
51 |
80 |
|||
75 |
44 |
65 |
23 |
36 |
|||
150 |
26 |
38 |
0 |
0 |
|||
300* |
11 |
16 |
0 |
0 |
|||
600* |
0 |
0 |
0 |
0 |
|||
1200* |
0 |
0 |
0 |
0 |
|||
2400* |
0 |
0 |
0 |
0 |
|||
a):1,1’- (1,1-dimethyl-3-methylene-1,3-propanediyl) bisbenzene treated group/negative control) x 100
*: White oily precipitations were noted at the time of application of the test solution and clean oily precipitations were noted on completion of the incubation.
The cell survival rate with the addition of S9 mix was 90% or more at 4.69-18.8 μg/mL, but it decreased with the increase in concentration at 37.5 μg/mL and higher, and no viable cells were observed at 600 μg/mL or higher.
The cell survival rate without the addition of S9 mix was 90% or more at 4.69-18.8 μg/mL, but it decreased with the increase in concentration at 37.5 μg/mL and higher, and no viable cells were observed at 150 μg/mL or higher
The 50% cell growth inhibition concentration (IC50) calculated by the Probit method were 101.5 μg/mL with S9 mix and 52.6 μg/mL without S9 mix.
A white oily precipitate was observed at the time test solution was added at concentrations of 300 μg/mL and higher both with and without S9 mix. A clear oily precipitate was observed at concentrations of 300 μg/mL and higher at the end of treatment.
Table 2: Cell growth inhibition test of 1,1’- (1,1-dimethyl-3-methylene-1,3-propanediyl) bisbenzene in cultured CHL cells –The continuous treatment method-
Test substance |
Concentration (µg/mL) |
Treated for 6 hr with S9 mix |
||
No of cells (x104/plate) |
Survival ratioa)(%) |
IC50 (µg/mL) |
||
Negative control (Dimethyl sulfoxide) |
- |
65 |
100 |
- |
1,1’- (1,1-dimethyl-3-methylene-1,3-propanediyl) bisbenzene |
4.69 |
62 |
95 |
23.9 |
9.38 |
59 |
91 |
||
18.8 |
44 |
68 |
||
37.5 |
23 |
35 |
||
75 |
0 |
0 |
||
150 |
0 |
0 |
||
300* |
0 |
0 |
||
600* |
0 |
0 |
||
1200* |
0 |
0 |
||
2400* |
0 |
0 |
||
a):1,1’- (1,1-dimethyl-3-methylene-1,3-propanediyl) bisbenzene treated group/negative control) x 100
*: White oily precipitations were noted at the time of application of the test solution and clean oily precipitations were noted on completion of the incubation.
The cell survival rate was 90% or more at 4.69 and 9.38 μg/mL, but it decreased with the increase in concentration at 18.8 μg/mL and higher, and no viable cells were observed at 75 μg/mL or higher.
IC50calculated by the Probit method was 23.9 μg/mL.
A white oily precipitate was observed at the time test solution was added at concentrations of 300 μg/mL and higher, and a clear oily precipitate was observed at concentrations of 300 μg/mL and higher at the end of treatment
Preliminary toxicity test
The dose range of the test item used in the preliminary toxicity test was 3.91 to 1001.6 μg/mL. The results for the Relative Suspension Growth (% RSG) were as follows:
Dose (µg/mL) |
% RSG (-S9) 4-Hour exposure |
% RSG (+S9) 4-Hour exposure |
% RSG (-S9) 24-Hour exposure |
0 |
100 |
100 |
100 |
9.22 |
45 |
64 |
18 |
18.44 |
1 |
33 |
5 |
36.88 |
0 |
1 |
0 |
73.75 |
0 |
1 |
0 |
147.5 |
0 |
0 |
0 |
295 |
0 |
0 |
0 |
590 |
0 |
0 |
0 |
1180 |
0 |
0 |
0 |
2360 |
0 |
0 |
0 |
Summary of Results
Experiment 1
4-Hours –S9 |
4-Hour +S9 |
||||||
Treatment (μg/mL) |
% RSG |
RTG |
MF§ |
Treatment (μg/mL) |
% RSG |
RTG |
MF§ |
0 |
100 |
1.00 |
115.68 |
0 |
100 |
1.00 |
136.05 |
2.5 Ø |
89 |
|
|
2.5 Ø |
94 |
|
|
5 Ø |
90 |
|
|
5 Ø |
89 |
|
|
7.5 |
87 |
1.00 |
107.64 |
10 |
75 |
0.71 |
158.23 |
10 |
83 |
0.93 |
99.35 |
15 |
58 |
0.58 |
139.56 |
12.5 |
64 |
0.71 |
113.49 |
20 |
53 |
0.50 |
163.78 |
15 |
53 |
0.68 |
117.86 |
25 |
53 |
0.48 |
160.24 |
17.5 |
31 |
0.31 |
131.52 |
30 |
40 |
0.41 |
153.60 |
20 |
12 |
0.12 |
177.80 |
35 |
28 |
0.24 |
170.89 |
Linear trend NS |
Linear trend NS |
||||||
EMS 400 |
63 |
0.52 |
1023.37 |
CP 2 |
66 |
0.36 |
1574.99 |
Experiment 2
4-Hours –S9 |
4-Hour +S9 |
||||||
Treatment (μg/mL) |
% RSG |
RTG |
MF§ |
Treatment (μg/mL) |
% RSG |
RTG |
MF§ |
0 |
100 |
1.00 |
144.47 |
0 |
100 |
1.00 |
131.32 |
2.5 |
93 |
0.89 |
142.19 |
5 Ø |
83 |
|
|
5 |
70 |
0.86 |
120.56 |
10 |
70 |
0.68 |
149.12 |
7.5 |
53 |
0.58 |
134.49 |
15 |
63 |
0.60 |
145.39 |
10 |
41 |
0.51 |
151.16 |
20 |
61 |
0.55 |
145.11 |
15 |
13 |
0.21 |
172.60 |
25 |
42 |
0.37 |
169.82 |
17.5 |
9 |
0.18 |
150.12 |
30 |
28 |
0.23 |
193.07 |
20 Ø |
7 |
|
|
35 X |
10 |
0.05 |
241.58 |
25 Ø |
3 |
|
|
40 Ø |
4 |
|
|
Linear trend NS |
Linear trend * |
||||||
EMS 400 |
48 |
0.39 |
1476.92 |
CP 2 |
57 |
0.30 |
1319.12 |
% RSG = Relative Suspension Growth
RTG = Relative Total Growth
MF§= 5- TFT resistant mutants/ 106 viable cells 2 days after treatment
Ø= Not plated for viability or 5 -TFT resistance
X= Treatment excluded from test statistics due to toxicity
NS= Not significant
* = p < 0.05
EMS = Ethylmethanesulphonate
CP = Cyclophosphamide
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Additional information from genetic toxicity in vitro:
Three in vitro genetic toxicity studies have been conducted on the test material, as follows:
in vitro gene mutation study in bacteria:A reverse mutation test was conducted in accordance with GLP and OECD Guideline 471 by the preincubation method using Salmonella typhimurium TA100, TA98, TA1535 and TA1537 and Escherichia coli WP2uvrA to examine its potential for inducing genetic mutations. The test was conducted with and without the addition of S9 mix. The number of revertant colonies at exposed to the test item at concentrations of 2.44-5000 μg/plate were less than twice the negative control in each strain with or without S9 mix in both main tests. From these results, the test item is judged to have no potential to induce genetic mutations under the conditions of this study.
in vitro chromosome aberration study: The potential for the test item to induce chromosomal aberrations was examined in accordance with GLP and OECD Guideline 473 using cultured mammalian cells (CHL/IU) by short treatment (6-hour treatment with and without S9 mix) and continuous treatment (24-hour treatment). Based on the 50 % cell growth inhibition concentrations and survival rate from the results of the cell growth inhibition test, five test concentrations of the test item were set in a common ratio of 2 at 12.5, 25, 50, 100 and 200 μg/mL for short treatment with S9 mix, 6.25, 12.5, 25, 50 and 100 μg/mL for short treatment without S9 mix, and 3.13, 6.25, 12.5, 25 and 50 μg/mL for continuous treatment (24-hour treatment). In the results of the test, the incidence of cells with numerical and structural aberrations was less than 5 % in both continuous treatment and short treatment. From these results, the test item was judged to have no chromosomal aberration inducibility under the conditions of this study.
in vitro gene mutation study in mammalian cells: The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line in accordance with GLP and OECD guideline 476. Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at 8 dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2 % S9 final concentration). In Experiment 2, the cells were treated with the test item at 8 dose levels using a 4-hour exposure group in the presence of metabolic activation (2 % S9 final concentration) and a 24-hour exposure group in the absence of metabolic activation. The dose range of test item was selected following the results of a preliminary toxicity test and for Experiment 1 was 2.5 to 20 μg/mL in the absence of metabolic activation, and 2.5 to 35 μg/mL in the presence of metabolic activation. The dose range for Experiment 2 was 2.5 to 25 μg/mL in the absence of metabolic activation, and 5 to 40 μg/mL in the presence of metabolic activation. The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. Precipitate of test item was not observed at any of the dose levels in the Mutagenicity Test. The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment. The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.
Justification for classification or non-classification
Three in vitro genetic toxicity studies have been conducted on the test material, an Ames study, a chromosome abberation study and a mouse lymphoma assay. All studies were conducted according to OECD guidelines and GLP and are adequately reported and therefore have been assigned a reliability 1.
The 3 in vitro genetic toxicity studies showed the test material to have no significant effects for gentoxicity. As such, the test material can be considered to be non-classified.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
