Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.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.

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Start: 29 October 2019, End: 29 November 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Constituent 1
Chemical structure
Reference substance name:
(8α,9R,8'''α,9'''R)-1,1'-[(2,3,5,6-tetrafluoro-1,4-phenylene)bis(methylene)]bis(6'-methoxycinchonan-1-ium-9-ol) dibromide
EC Number:
857-673-6
Cas Number:
1879067-61-4
Molecular formula:
C48H52F4N4O4.2Br
IUPAC Name:
(8α,9R,8'''α,9'''R)-1,1'-[(2,3,5,6-tetrafluoro-1,4-phenylene)bis(methylene)]bis(6'-methoxycinchonan-1-ium-9-ol) dibromide
Test material form:
solid: particulate/powder

Method

Target gene:
functionally hemizygous hypoxanthine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO-K1) cells
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CHO-K1 cells were obtained from the European Collection of Cell Cultures. Cells were stored at -196 to -150 °C, in heat-inactivated foetal calf serum (HiFCS) containing 10% dimethyl sulphoxide (DMSO). The cells are screened periodically for the absence of mycoplasma contamination. The modal chromosome number is 20 and the cell doubling time is ca. 12 hours.
The following media were used: H0 = Ham’s Nutrient Mixture F12 (containing 1 mM L glutamine), supplemented with 50 ng/mL amphotericin B / 20 IU/mL penicillin / 20 μg/mL streptomycin; H10 = H0 medium supplemented with 10% heat inactivated fetal calf serum
The selective medium, in which only HPRT deficient cells will grow, consisted of H10 supplemented with 6-TG at a final concentration of 10 µg/mL. All cell cultures were maintained at 34 to 39 °C in an humidified atmosphere of 5% CO2 in air.
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction was prepared from male Sprague-Dawley derived rats, dosed with phenobarbital/5,6-benzoflavone to stimulate mixed-function oxidases in the liver. S9 mix contained: S9 fraction (10% v/v), glucose-6-phosphate (6.9 mM), NADP (1.4 mM) in H0.
Test concentrations with justification for top dose:
Preliminary toxicity test: 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL; No precipitate was observed by eye at the end of treatment at concentrations of up to 2000 µg/mL and this was the highest concentration plated for determination of relative survival (RS) in both the absence and presence of S9 mix.
Main experiment (without S9 mix, 3 hours): 9.5, 95, 475, 950, 1100, 1250, 1400, 1550, 1700, 1850 and 2000 µg/mL (cultures assessed for determination of the mutant phenotype were 9.5, 1100, 1250, 1550, 1700, 1850 and 2000 µg/mL); Precipitate was seen by eye at the end of treatment at 2000 µg/mL.
Main experiment (with S9 mix, 3 hours): 9.5, 95, 475, 950, 1100, 1250, 1400, 1550, 1700, 1850 and 2000 µg/mL (cultures assessed for determination of the mutant phenotype were 9.5, 95, 475, 950, 1100, 1250 and 1400 µg/mL); Precipitate was observed by eye at the end of treatment at concentrations of 1400 µg/mL and above. Therefore 1400 µg/mL was the maximum concentration continued.
Vehicle / solvent:
Dimethyl sulfoxide (1% v/v)
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
ethylmethanesulphonate
Details on test system and experimental conditions:
Preliminary toxicity test: A cell suspension was prepared at 3 x 10E+05 cells/mL. Aliquots of 20 mL of this suspension were dispensed into 150 cm2 flasks, one flask per concentration of test item and two for the vehicle controls both in the absence and in the presence of S9 mix. Eight concentrations of the test item were used. Two additional flasks were prepared to determine average cell density across all flasks at the beginning of the treatment period; this was subsequently used to calculate the adjusted cloning efficiency. The cells were incubated for approximately 24 hours at between 34 and 39 °C, in an atmosphere of 5% CO2 in air, prior to exposure to the test item on Day 1. Prior to treatment, the medium was replaced with 16 mL of fresh medium and aliquots of 4 mL of H0 or S9 mix as appropriate followed by 200 µL of test item (at 100 times the desired final concentration) or vehicle. The flasks were returned to the incubator for a further three hours.
At the end of the exposure period the cells were harvested. Samples were taken from each culture, the cells counted and the cell density calculated. Three flasks per culture were seeded with 200 cells each (survival flasks). The plates were incubated at between 34 and 39 °C, in a humidified atmosphere of 5% CO2 in air, for seven days, after which time colonies growing in the plate were fixed and stained in a methanol:Giemsa solution (4:1 v/v). Colonies were counted and the Day 1 relative survival was calculated.

Main mutation test: The procedure for the main experiments was the same as that for the preliminary experiments, with the following exceptions: positive control cultures were included for all experiments; duplicate cultures were prepared for all cultures (quadruplicate cultures for vehicle controls); and following preparation of survival flasks, 2 x 10E+06 cells from each culture were seeded into 150 cm2 flasks containing 30 mL H10 and incubated for seven days to allow expression of the mutant phenotype. The cultures were sub-cultured during the expression period and after a total of seven days, were harvested. For each culture, three flasks were seeded with 200 cells each, to determine cloning efficiency and five flasks with 5 x 10E+05 cells each in selective medium to determine cloning efficiency. The flasks were returned to the incubator for approximately seven days at between 34 and 39 °C in a humidified atmosphere of 5% CO2 in air. At the end of this incubation period, colonies growing in the flasks were fixed and stained in a methanol:Giemsa solution (4:1 v/v) and counted.
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a statistically significant increase in mean mutant frequency compared with the concurrent negative control
b) the increase in mean mutant frequency is concentration-related when evaluated with an appropriate trend test
c) any of the results (mean mutant frequency) are outside the distribution of the historical negative control data (above the upper 95% confidence limit)
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
a) none of the test concentrations exhibits a statistically significant increase in mean mutant frequency compared with the concurrent negative control
b) there is no concentration-related increase in mean mutant frequency when evaluated with an appropriate trend test
c) all results (mean mutant frequency) are inside the distribution of the historical negative control data (within the 95% confidence limits).
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
Statistics:
The statistical significance of the data was analysed by weighted analysis of variance, weighting assuming a Poisson distribution following the methods described by Arlett et al. (1989). Tests were conducted for a linear concentration-response relationship of the test item, for non-linearity and for the comparison of positive control and treated groups to vehicle control.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary toxicity test: The osmolality and pH of the substance in medium were measured by analysing samples of H10 media, dosed at 1% (v/v), with either the vehicle (DMSO) or a test substance formulation at 200 mg/mL (to give a final concentration of 2000 μg/mL). No fluctuations in osmolality of the medium of more than 50 mOsmol/kg and no fluctuations in pH of more than 1.0 unit were observed for this formulation compared with the vehicle control. The maximum final concentration tested in the preliminary toxicity test was 2000 μg/mL as this is the standard limit concentration within this test system.
Prior to treatment with the substance, cultures established concurrently to those used in this experiment were assessed to ascertain the cell density. The cell concentration was confirmed to be 1.2 x 10E+06 cells/mL (i.e. 12 x 10E+06 cells treated per concentration, 24 x 10E+06 cells for the vehicle control). This cell concentration was used in the calculation of the adjusted cloning efficiency.
The substance was initially dosed at concentrations up to 2000 µg/mL. No precipitate was observed by eye at the end of treatment at concentrations of up to 2000 µg/mL and this was the highest concentration plated for determination of relative survival (RS) in both the absence and presence of S9 mix. Exposure to the substance for 3 hours at concentrations from 15.63 to 2000 µg/mL in both the absence and presence of S9 mix resulted in RS values from 80% and 4% and from 93% and 1% respectively.

Main Mutation Test - 3-hour Treatment in the Absence of S9 Mix: Prior to treatment, cultures established concurrently to those used in this experiment were assessed to ascertain the cell density. The cell concentration was confirmed to be 1.5 x 10E+06 cells/mL (i.e. 30 x 10E+06 cells treated per concentration, 60 x 10E+06 cells for the vehicle control). This cell concentration was used in the calculation of the adjusted cloning efficiency.
Cultures were exposed to the substance at concentrations from 9.5 to 2000 µg/mL. Precipitate was seen by eye at the end of treatment at 2000 µg/mL. Exposure to the substance resulted in mean RS values from 101 to 28%. Cultures treated at 9.5, 1100, 1250, 1550, 1700, 1850 and 2000 µg/mL were selected for expression of mutant frequency. No significant increases in mutant frequency were observed after exposure. None of the treated groups induced mean mutant frequencies above the laboratory historical 95% confidence limits and tests for both a linear trend and non-linearity were applied across all treatment groups, neither of which was statistically significant. Therefore, this experiment is concluded to be clearly negative.
EMS, the positive control, induced a significant increase in mean mutant frequency compatible with the laboratory’s historical positive control data demonstrating the sensitivity of the test system.

Main Mutation Test - 3-hour Treatment in the Presence of S9 Mix: Prior to treatment, cultures established concurrently to those used in this experiment were assessed to ascertain the cell density. The cell concentration was confirmed to be 1.5 x 10E+06 cells/mL (i.e. 30 x 10E+06 cells treated per concentration, 60 x 10E+06 cells for the vehicle control). This cell concentration was used in the calculation of the adjusted cloning efficiency.
Cultures were exposed to the substance at concentrations from 9.5 to 2000 µg/mL. Precipitate was observed by eye at the end of treatment at concentrations of 1400 µg/mL and above. Therefore 1400 µg/mL was the maximum concentration continued. Exposure to the substance resulted in mean RS values from 88 to 62%. Cultures treated at 9.5, 95, 475, 950 1100, 1250 and 1400 µg/mL were selected for expression of mutant frequency. No significant increases in mutant frequency were observed after exposure. None of the treated groups induced mean mutant frequencies above the laboratory historical 95% confidence limits and tests for both a linear trend and non-linearity were applied across all treatment groups, neither of which was statistically significant. Therefore, this experiment is concluded to be clearly negative.
3MC, the positive control, induced a significant increase in mean mutant frequency compatible with the laboratory’s historical positive control data demonstrating the sensitivity of the test system and efficacy of the S9 metabolic fraction.

Any other information on results incl. tables

Summary of results








































































































































































  3-hour exposure, without S9 mix  3-hour exposure, with S9 mix  
Test itemConc (µg/mL)Mean relative survival (%)Mean mutant frequency (per 10E+06 viable cells)95% conf. intervalsMean relative survival (%)Mean mutant frequency (per 10E+06 viable cells)95% conf. intervals
DMSO solvent010010.731.4 -15.41009.420.0 - 15.7
Test subst.9.59512.35 869.12 
 9585NA 886.89 
 47583NA 859.54 
 95049NA 628.36 
 11008012.33 8010.74 
 12507013.89 685.66 
 1400101NA 77 b8.11 
 1550608.91 NA cNA 
 17005110.48 NA cNA 
 1850457.04 NA cNA 
 200028 b9.35 NA cNA 
Ethyl methanesulphonate2508288.73***38.9 - 128.9Not testedNot tested 
3-methylcholanthrene5Not testedNot tested 8279.23***24.0 - 87.7

b) precipitate observed at the end of treatment


c) precipitate observed at the end of treatment, cultured discarded


*** p<0.001; all other cultures p >0.05; treated groups were compared to the vehicle control using one-tailed Dunnett's tests for an increase and the positive control was compared to the vehicle control using a one-tailed t-test for an increase


Applicant's summary and conclusion

Conclusions:
The substance was not mutagenic in this valid in vitro mammalian cell mutation study.
Executive summary:

The substance was tested under GLP for mutagenic potential in an in vitro mammalian cell mutation assay to OECD TG 476. This test system is based on detection and quantitation of forward mutation at the functionally hemizygous hypoxanthine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO-K1) cells. Two independent tests, one in the absence of exogenous metabolic activation (S9 mix) and one in the presence of S9 mix, were conducted.
The vehicle was dimethyl sulfoxide (DMSO), in which the test item dissolved at up to 200 mg/mL. The highest final concentration used in the preliminary toxicity experiment was standard limit concentration 2000 µg/mL recommended in the current guideline. No precipitate was observed by eye at the end of treatment at up to 2000 µg/mL. Cytotoxicity was measured as Day 1 relative survival (RS). After exposure to the substance at concentrations from 15.63 to 2000 µg/mL RS values ranged from 80 to 4% and from 93 to 1%, in the absence and presence of S9 mix respectively.
In the main mutation experiment in the absence of S9 mix, cells were exposed to concentrations from 9.5 to 2000 µg/mL. Precipitate was observed by eye at the end of treatment at 2000 µg/mL. Mean RS values ranged from 101 to 28% relative to the vehicle control. The substance did not induce a statistically significant increase in mean mutant frequency. None of the treated groups induced mean mutant frequencies above the laboratory historical 95% confidence limits and tests for both a linear trend and non-linearity were applied across all treatment groups, neither of which was statistically significant. The positive control, ethyl methanesulphonate, induced a significant increase in mean mutant frequency demonstrating the correct functioning of the assay. The criteria for a clearly negative response were therefore met in this treatment.
In the main mutation experiment in the presence of S9 mix, cells were exposed to concentrations from 9.5 to 2000 µg/mL. Precipitate was observed by eye at the end of treatment at concentrations of 1400 µg/mL and above. Therefore 1400 µg/mL was the maximum concentration continued. Mean RS values ranged from 88 to 62% relative to the vehicle control. The substance did not induce a statistically significant increase in mean mutant frequency. None of the treated groups induced mean mutant frequencies above the laboratory historical 95% confidence limits and tests for both a linear trend and non-linearity were applied across all treatment groups, neither of which was statistically significant. The positive control, 3-methylcholanthrene, induced a significant increase in mean mutant frequency demonstrating the correct functioning of the assay and the efficacy of the S9 metabolic fraction. The criteria for a clearly negative response were therefore met in this treatment