Reaction products of tetrabromophthalic anhydride with 2,2'-oxydiethanol and methyloxirane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 August 2020 - 11 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

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:

Adopted 29 July 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

Test material information

1

Reference substance name:

Reaction products of 3,4,5,6-tetrabromobenzene-1,2-dicarboxylate with 2,2'-oxy-diethanol and 2-epoxypropane

EC Number:

616-436-5

Cas Number:

77098-07-8

Molecular formula:

Ester product of C8 H2 Br4 O4 with C4 H10 O3 and C3 H8 O2 and condensation products, thereof.

IUPAC Name:

Reaction products of 3,4,5,6-tetrabromobenzene-1,2-dicarboxylate with 2,2'-oxy-diethanol and 2-epoxypropane

Test material form:

liquid: viscous

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: The V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. The high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50%) make it an appropriate cell line to use for this study type.

For cell lines:
- Absence of Mycoplasma contamination: Master stocks of cells were tested and found to be free of mycoplasma.
- Methods for maintenance in cell culture: The stock of cells is stored in liquid nitrogen. For use, a sample of cells will be removed before the start of the study and grown in Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% fetal bovine serum (FBS)) at approximately 37 °C with 5% CO2 in humidified air.
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: The cells have a stable karyotype.
- Periodically ‘cleansed’ of spontaneous mutants: yes

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: The S9 Microsomal Enzyme Fraction was purchased from Moltox and Lot no 4272 with the expiry date of 16 July 2022, and Lot no 4370 with the expiry date of 24 November 2022, were used in this study.
- method of preparation of S9 mix: The S9 mix was prepared by mixing S9 with a phosphate buffer containing NADP (5 mM), G6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20% or 10% S9 concentration.
- concentration or volume of S9 mix and S9 in the final culture medium: The final concentration of S9 when dosed at a 10% volume of S9-mix was 2% for the Preliminary Toxicity Test and the Main Experiment.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): checked, with quality control certificate

Test concentrations with justification for top dose:

The concentrations used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by test item induced toxicity, as recommended by the OECD 476 guideline.

Main experiment:
4-hour without S9
0, 37.5, 43.75, 50, 56.25, 62.5, 68.75, 75 µg/mL
4-hour with S9 (2%)
0, 70, 80, 90, 100, 110, 120, 130 µg/mL

Due to optimum levels of toxicity not being achieved in the absence of metabolic activation, a repeat of this exposure group was performed with adjusted dose levels. The concentrations of test item plated for relative survival, cloning efficiency, and expression of mutant colonies were as follows:

Main Experiment Repeat:
4-hour without S9
0, 12.5, 25, 50, 60, 70 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone

- Justification for choice of solvent/vehicle: The test item was found to be fully miscible in acetone at 1000 mg/mL. Acetone was also the sponsors preferred solvent vehicle. A GLP compliant study performed outside the confines of this study showing homogeneity and stability in acetone is available via Eurofins EAG Agroscience, LLC study number 471C-157.

- Justification for percentage of solvent in the final culture medium: Acetone is toxic to V79 cells at dose volumes greater than 0.5% of the total culture volume. Therefore, the test item was formulated at 1000 mg/ml and dosed at 0.5% to give a maximum concentration of 5000 μg/mL.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other:

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 3 (2 without S9,1 with S9)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Cells were seeded at 1 x 10E7 cells/225 cm2 flask approximately 24 hours being exposed to the test or control items. This was demonstrated to provide at least 20 x 10E6 available for dosing in each flask using a parallel flask.
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7d
- Selection time (if incubation with a selective agent): 7 d
- Fixation time (start of exposure up to fixation or harvest of cells): 14d + 4 h
- Method used: Fixation and staining of all flasks/petri dishes was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure: 11 μg/mL 6-Thioguanine (6-TG), incubated for 7 days at 37 °C in an incubator with humidified atmosphere of 5% CO2 in air.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2 x 10E5 cells/petri dish (ten replicates per group). Mutant colonies were manually counted and recorded for each dish.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: cloning efficiency; relative survival (RS)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
mutant frequency/10E6 survival rate (MFS 10E-6)

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly positive if, in any of the experimental conditions examined:
i) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent solvent control.
ii) The increase is considered to be concentration-related when evaluated with a trend test.
iii) The results for the test item concentrations are outside the range of the historical solvent control data and 95% control limits.
When all these criteria are met, the test item is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in all of the experimental conditions examined:
i) None of the test concentrations exhibits a statistically significant increase compared with the concurrent solvent control.
ii) There is no concentration related increase when evaluated with a trend test.
iii) The results for the test item concentrations are within the range of the historical solvent control data and 95% control limits.
The test item is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
There is no requirement for verification of a clearly positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgment and/or further investigations. Performing a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, S9 concentration, and exposure time) may be useful.

Statistics:

When there is no indication of any increases in mutant frequency at any concentration then statistical analysis may not be necessary. In all other circumstances the mutant frequency was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on numbers of mutant colonies. A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the mutant frequency was less than 0.05 and there was a dose-related increase.
The dose-relationship (trend-test) was assessed using a linear regression model. An arcsin square-root transformation was applied to the mutant frequency (excluding positive controls). A linear regression model was then applied to these transformed values with dose values fitted as the explanatory variable. The F-value from the model was assessed at the 5% statistical significance level.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: please refer to 'Any other information on results'
- Data on osmolality: please refer to 'Any other information on results'
- Precipitation and time of the determination: At the end of the exposure period, precipitate of the test item was observed at 150 μg/mL in the absence of metabolic activation. In the Repeat Main Experiment no precipitation was observed.

RANGE-FINDING/SCREENING STUDIES (if applicable):
The preliminary cytotoxicity test was performed on cell cultures plated out at 1 x 10E7 cells/225 cm2 flask approximately 24 hours before dosing. This was demonstrated to provide at least 20 x 10E6 available for dosing in each flask using a parallel flask, counted at the time of dosing. On dosing, the growth media was removed and replaced with serum-free Minimal Essential Medium (MEM). One flask per concentration was treated for 4-hours without metabolic activation and for 4-hours with metabolic activation (2% S9). Due to the precipitate observed in the solubility check, the concentrations of test item used was 0, 4.69, 9.38, 18.75, 37.5, 75, 150, 300, 600, and 1200 μg/mL.
Exposure was for 4 hours at approximately 37 °C with a humidified atmosphere of 5% CO2 in air, after which the cultures were washed twice with phosphate buffered saline (PBS) before being detached from the flasks using trypsin. Cells from each flask were suspended in MEM with 10% FBS, a sample was removed from each concentration group and counted using a Coulter counter. For each culture, 200 cells were plated out into three 25 cm2 flasks with 5 mL of MEM with 10% FBS and incubated for 6 days at approximately 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air. The cells were then fixed and stained and total numbers of colonies in each flask counted to give relative survival (RS). A comparison of the test item to vehicle control relative survivals gave the relative toxicity of each test item concentration.
Results from the preliminary cytotoxicity test were used to select the test item concentrations for the mutagenicity experiment.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Please refer to attached tables

For all test methods and criteria for data analysis and interpretation:
Please refer to attached tables

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
Please refer to attached tables

- Genotoxicity results:
Please refer to attached tables

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
Please refer to attached tables

Any other information on results incl. tables

The pH and osmolality readings are presented in the following table:

Concentration [µg/mL]	0	19.53	39.06	78.13	156.25	312.5	625	1250	2500	5000
pH	7.53	7.53	7.53	7.53	7.54	7.54	7.54	7.53	7.60	7.61
mOsm	384	370	-	370	387	392	385	383	-	336

Applicant's summary and conclusion

Conclusions:

The test item did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation. It was, therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.

Executive summary:

The purpose of this study is to assess the potential mutagenicity of the test item on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line. The GLP-compliant study was performed according to OECD Guideline 476.
Chinese hamster (V79) cells were treated with the test item at up to ten concentrations, in duplicate, together with solvent (acetone) and positive controls in the absence and presence of metabolic activation (S9).
The concentrations used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by test item induced toxicity, as recommended by the OECD 476 guideline. The concentrations of test item plated for relative survival, cloning efficiency, and expression of mutant colonies were as follows:
Main Experiment
4-hour without S9
0, 37.5, 43.75, 50, 56.25, 62.5, 68.75, 75 µg/mL
4-hour with S9 (2%)
0, 70, 80, 90, 100, 110, 120, 130 µg/mL
Due to optimum levels of toxicity not being achieved in the absence of metabolic activation, a repeat of this exposure group was performed with adjusted dose levels. The concentrations of test item plated for relative survival, cloning efficiency, and expression of mutant colonies were as follows:
Main Experiment Repeat
4-hour without S9
0, 12.5, 25, 50, 60, 70 µg/mL

The solvent (acetone) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus.
The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system.
The test item did not induce any statistically significant increases in mutant frequency at any of the concentrations in the presence of metabolic activation. There were also no statistically significant concentration related increases when evaluated with a trend test, and all of the values observed were within the historical control range and 95% control limits for solvent controls.
The test item induced very small but statistically significant increases in mutant frequency in the absence of metabolic activation in the repeat experiment (MFS 15 p=0.010** at 50 mg/mL and MFS 12 p=0.018* at 60 mg/mL). However, no concentration relation was noted when evaluated with a trend test, and all the values observed were within the historical control range and 95% control limits for solvent controls (5.81 to 19.61 in the absence of S9 and 5.90 to 19.02 in the presence of S9). The response was therefore considered to be spurious and of no toxicological significance.
The results observed in both the absence and presence of metabolic activation were considered to fulfill the criteria for a clearly negative outcome.
Conclusion: The test item did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation. Saytex RB-79 was, therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.