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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria, OECD 471 (Ames test), GLP, S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strain WP2uvrA, +/-S9, plate incorporation method and pre-incubation method: negative

Gene mutation in mammalian cells, OECD 476 (HPRT test), GLP, V79 cells, +/-S9: negative

Chromosome aberration in mammalian cells, OECD 473, GLP, human lymphocytes, +/-S9: negative

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

HPRT

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.

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.

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

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 AUG 2020 - 9 JUL 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environmental (MOE) Guidelines of 21 December 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: 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.
- Normal cell cycle time (negative control): average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours

For lymphocytes:
- Sex, age and number of blood donors:
Preliminary Toxicity Test: female, aged 24 years
Preliminary Toxicity Test Repeat 24-hour only: male, aged 30 years
Main Experiment: female, aged 28 years
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: not pooled
- Mitogen used for lymphocytes: The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % fetal bovine serum (FBS), at approximately 37ºC with 5 % CO2 in humidified air.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: purchased from Moltox, Lot No. 4222, Expiry 12 March 2022
- method of preparation of S9 mix: 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).
- 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 into culture media, was 2%.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): yes, incl. certificate

Test concentrations with justification for top dose:

4(20)-hour without S9: 0, 5, 10, 20, 40, 60, 80, 120, 160 µg/mL
4(20)-hour with S9 (2%): 0, 5, 10, 20, 40, 60, 80, 120, 160 µg/mL
24-hour without S9: 0, 5, 10, 20, 30, 40, 60, 80, 160 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 human lymphocytes 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 the maximum recommended dose level of 5000 μg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): quadruplicate (vehicle control), duplicate (dose groups)
- Number of independent experiments: 3

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment, Harvest time after the end of treatment (sampling/recovery times):
i) 4-hour exposure to the test item without S9-mix, followed by 20-hour recovery period in treatment-free media prior to cell harvest. The dose range of test item used was 5 to 160 μg/mL.
ii) 4-hour exposure to the test item with S9-mix(2%), followed by 20-hour recovery period in treatment-free media prior to cell harvest. The dose range of test item used was 5 to 160 μg/mL.
i) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 5 to 160 μg/mL.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure: Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/mL) two hours before the required harvest time.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): 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.
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.
When the slides were dry, they were stained in 5 % Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): Where possible 1000 cells per culture were evaluated for the incidence of metaphase cells and expressed as the mitotic index and as a percentage of the vehicle control value.
Where possible, 300 consecutive well-spread metaphases from each concentration were counted, 600 from the vehicle control (150 per replicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): 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). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
- Determination of polyploidy, Determination of endoreplication: In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) (including the incidence of cells with endoreduplicated chromosomes) was also reported.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI)

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.

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in any of the experimental conditions examined:
1) The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
3) There is no concentration-related increase at any dose level.
A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related.
When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include numerical aberrations in the form of polyploidy and endoreduplicated cells.

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.
If required, the concentration relationship will be assessed using a linear regression model. An arcsine square root transformation will be applied to the frequency of cells with aberrations excluding gaps (not including positive control(s)). A linear regression model will be applied to these transformed values with concentration values fitted as the explanatory variable. The F-value from the model will be assessed at the 5% statistical significance level.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: precipitation in the 4 h exposure group, toxicity in the 24 h exposure group

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: please refer to attached tables

RANGE-FINDING/SCREENING STUDIES (if applicable):
The dose range for the Cell Growth Inhibition Test was initially 19.53 to 5000 μg/mL for all three exposure groups, which was the maximum recommended dose level. However due to excessive toxicity seen in the 24-hour continuous cultures the Cell Growth Inhibition Test was repeated (24 hours only). Therefore, the maximum dose for the 24-hours continuous exposure was limited by test item-induced toxicity, the repeat dose range was 0.313 to 80 μg/mL.
Precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure period at and above 78.13 μg/mL in the 4-hour exposure groups in the absence and presence of metabolic activation. In the 24-hour continuous exposure precipitate was observed at and about 156.25 μg/mL but precipitate was not observed in the repeat Cell Inhibition Test due to the lower dose concentrations.
Hemolysis was observed following exposure to the test item at and above 78.13 μg/mL in the all three exposure groups and at 80 μg/mL in the repeat 24-hour exposure group in the absence of metabolic activation. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present at up to 39.06 μg/mL in the 4-hour exposure groups in the absence and presence of metabolic activation, and at up to 80 μg/mL in the 24-hour exposure group in the absence of metabolic activation. The test item induced evidence of marked toxicity in all three of the exposure groups.
The selection of the maximum dose level for the Main Experiment was based on the lowest precipitating dose level in the 4-hour exposure groups in both the absence and presence of metabolic activation, and limited by test item-induced toxicity in the 24-hour exposure group in the absence of metabolic activation.

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

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For lymphocytes in primary cultures: mitotic index (MI)
please refer to attached tables
- Genotoxicity results (for both cell lines and lymphocytes)
o Definition for chromosome aberrations, including gaps
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excluding gaps
o Changes in ploidy (polyploidy cells and cells with endoreduplicated chromosomes) if seen
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)
- Positive historical control data, Negative (solvent/vehicle) historical control data: please refer to attached 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

Conclusions:

The test item was considered to be non-clastogenic to human lymphocytes in vitro both in the presence and absence of a metabolic activation system (rat liver S9 mix).

Executive summary:

The purpose of this study was the detection of structural chromosomal aberrations in cultured mammalian cells. The GLP-compliant study was performed according to OECD Guideline 473.
Duplicate cultures of human lymphocytes, treated with the test item, and quadruplicate cultures for the solvent controls, were evaluated for chromosome aberrations at up to four dose levels, together with solvent 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 Cell Growth Inhibition Test (Preliminary Toxicity Test) where the results indicated that the maximum concentration should be limited by the onset of test item precipitate in the 4-hour exposure groups in both the absence and presence of metabolic activation, and test item-induced toxicity in the 24-hour exposure group in the absence of metabolic activation. The dose levels selected for the Main Test were as follows:

4(20)-hour without S9
0, 5, 10, 20, 40, 60, 80, 120, 160 µg/mL
4(20)-hour with S9 (2%)
0, 5, 10, 20, 40, 60, 80, 120, 160 µg/mL
24-hour without S9
0, 5, 10, 20, 30, 40, 60, 80, 160 µg/mL

All solvent (acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.
All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level in the 4-hour exposure group in the absence of metabolic activation, and a dose range that achieved optimum levels of test item-induced toxicity in the 24-hour exposure group in the absence of metabolic activation.
In the presence of metabolic activation there was a small but statistically significant increase compared to the concurrent control in the frequency of cells with aberrations at 80 μg/mL (lowest precipitating dose level). The response at 80 μg/mL was only break type aberrations (1.7 % ctb), which was well within the laboratory historical control range (0 - 2 %) for a vehicle, the vehicle on this occasion was also very low, therefore it was considered to be an artifactual response which was within normal variation and of no toxicological relevance. There was no concentration related response demonstrated when evaluated with the trend test. The P value from the trend test was 0.162 and was therefore not statically significant.
As a conclusion, the test item, was considered to be non-clastogenic to human lymphocytes in vitro both in the presence and absence of a metabolic activation system (rat liver S9 mix).

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 AUG 2020 - 13 JUL 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

As the result of Experiment 1 (see cross-reference) was considered negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation (S9-mix).

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997 as corrected in 2020

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines of 31 March 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine (Salmonella strains), tryptophan (E.coli)

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:

Type and composition of metabolic activation system:
- source of S9: The Phenobarbitone / β-Naphthoflavone induced S9 Microsomal fractions (Sprague-Dawley) used in this study were purchased from Moltox; Lot No. 4222 and the protein level was adjusted to 20 mg/mL.
- method of preparation of S9 mix: The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
S9 fraction 5.0 mL
1.65 M KCl/0.4 M MgCl2 1.0 mL
0.1 M Glucose-6-phosphate 2.5 mL
0.1 M NADP 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL
Sterile distilled water 14.5 mL
- concentration or volume of S9 mix and S9 in the final culture medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment. A S9 Quality Control and Production Certificate is available.

Test concentrations with justification for top dose:

The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.
Seven test item concentrations were selected in Experiment 2 in order to ensure the study achieved at least four non-toxic dose levels as required by the test guideline, and were selected based on the lack of cytotoxicity noted in Experiment 1 and the potential for a change in the cytotoxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration and acetone at 100 mg/mL in solubility checks performed in-house. After consultation with the Sponsor, acetone was selected as the 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 the bacterial cells at 0.1 mL (100 μL) after employing the pre-incubation modification; therefore all of the formulations for Experiment 2 were prepared at concentrations two times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 0.05 mL (50 μL) aliquots.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene; 4-Nitroquinoline-1-oxide

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: 2 (Experiment 1 is linked under 'Cross-reference')

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 min (with shaking)
- Exposure duration/duration of treatment: 48 - 72 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning of the background bacterial lawn (toxicity). Sporadic manual counts were required at 5000 μg/plate because of a test item film. Additional manual counts were also performed due to spreading colonies which prevented an accurate automated count.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response .
5. Statistical analysis of data as determined by UKEMS.
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments give clear positive or negative results, in some instances the data generated prohibit making a definite judgment about test item activity. Results of this type are reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnett’s Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity observed in the absence of S9 from 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity observed in the absence of S9 from 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity observed in the absence of S9 from 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity observed in the absence of S9 from 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: A test item film (white and cloudy in appearance) was noted at 5000 μg/plate in both the presence and absence of metabolic activation (S9-mix). This observation did not prevent the scoring of revertant colonies.

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:
- Concentration-response relationship where possible
- Statistical analysis; p-value if any
- Any other criteria: e.g. GEF for MLA
please refer to attached tables

Ames test:
- Signs of toxicity: The test item induced a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains in the absence of metabolic activation (S9-mix) only from 1500 μg/plate for TA100, TA98 and TA1537 and at 5000 μg/plate for TA1535. No toxicity was noted for Escherichia coli strain WP2uvrA dosed in either the absence or presence of S9 mix or any of the Salmonella strains dosed in the presence of S9-mix.
- Individual plate counts: please refer to attached tables
- Mean number of revertant colonies per plate and standard deviation: 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)
- Positive historical control data, Negative (solvent/vehicle) historical control data: please refer to attached tables

Conclusions:

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test the test item was considered to be non-mutagenic.

Executive summary:

An Ames Test according to OECD Guideline 471 and GLP was performed. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 (plate incorporation) was based on OECD TG 471 and was 1.5 to 5000 μg/plate. The experiment was performed on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 5 to 5000 μg/plate. Seven test item concentrations were selected in Experiment 2 in order to ensure the study achieved at least four non-toxic dose levels as required by the test guideline, and were selected based on the lack of cytotoxicity noted in Experiment 1 and the potential for a change in the cytotoxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.
The vehicle (acetone) control plates gave counts of revertant colonies within the normal range of the historical control data. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation and were within the control range for the different tester strains and positive control substances. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method).
Based on the results of Experiment 1, the same maximum dose level (5000 μg/plate) was employed in the second mutation test (pre-incubation method). The test item induced a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains, initially from 1500 μg/plate in the absence of metabolic activation (S9-mix). No toxicity was noted for Escherichia coli strain WP2uvrA dosed in either the absence or presence of S9-mix or any of the Salmonella strains dosed in the presence of S9-mix. A test item film (white and cloudy in appearance) was noted at 5000 μg/plate in both the presence and absence of metabolic activation (S9-mix) in Experiments 1 and 2. This observation did not prevent the scoring of revertant colonies.
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method).
Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). One statistically significant value was noted (TA100 at 50 μg/plate in the absence of metabolic activation (S9-mix). However, as the maximum fold increase was only 1.3 times the concurrent vehicle control and there was no evidence of a dose-related response or reproducibility, the response was considered an accidental finding reflecting biological variance and not being of toxicological relevance. Therefore, this value has not been highlighted in Table 4 as it did not meet the required criteria for a positive response.
In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test the test item was considered to be non-mutagenic.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 AUG 2020 - 13 JUL 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997 as corrected in 2020

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines of 31 March 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine (Salmonella strains), tryptophan (E.coli)

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:

Type and composition of metabolic activation system:
- source of S9: The Phenobarbitone / β-Naphthoflavone induced S9 Microsomal fractions (Sprague-Dawley) used in this study were purchased from Moltox; Lot No. 4222 and the protein level was adjusted to 20 mg/mL.
- method of preparation of S9 mix: The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
S9 fraction 5.0 mL
1.65 M KCl/0.4 M MgCl2 1.0 mL
0.1 M Glucose-6-phosphate 2.5 mL
0.1 M NADP 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL
Sterile distilled water 14.5 mL
- concentration or volume of S9 mix and S9 in the final culture medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment. A S9 Quality Control and Production Certificate is available.

Test concentrations with justification for top dose:

The maximum concentration was 5000 μg/plate (the OECD TG 471 maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration and acetone at 100 mg/mL in solubility checks performed in-house. After consultation with the Sponsor, acetone was selected as the 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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene; 4-Nitroquinoline-1-oxide

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: 2 (experiment 2 is found as cross-reference)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 - 72 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Plates are scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning of the background bacterial lawn (toxicity). Sporadic manual counts were performed due to spreading colonies which prevented an accurate automated count.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response .
5. Statistical analysis of data as determined by UKEMS.
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments give clear positive or negative results, in some instances the data generated prohibit making a definite judgment about test item activity. Results of this type are reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnett’s Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: No
- Other confounding effects: A test item film (white and cloudy in appearance) was noted at 5000 μg/plate in both the presence and absence of metabolic activation (S9-mix). This observation did not prevent the scoring of revertant colonies.

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:
- Concentration-response relationship where possible
- Statistical analysis; p-value if any
- Any other criteria: e.g. GEF for MLA
Please refer to attached tables

Ames test:
- Signs of toxicity: There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).
- Individual plate counts: Please refer to attached tables
- Mean number of revertant colonies per plate and standard deviation: 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)
- Positive historical control data, Negative (solvent/vehicle) historical control data: Please refer to attached tables

Conclusions:

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test the test item was considered to be non-mutagenic.

Executive summary:

An Ames Test according to OECD Guideline 471 and GLP was performed. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 (plate incorporation) was based on OECD TG 471 and was 1.5 to 5000 μg/plate. The experiment was performed on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 5 to 5000 μg/plate. Seven test item concentrations were selected in Experiment 2 in order to ensure the study achieved at least four non-toxic dose levels as required by the test guideline, and were selected based on the lack of cytotoxicity noted in Experiment 1 and the potential for a change in the cytotoxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.
The vehicle (acetone) control plates gave counts of revertant colonies within the normal range of the historical control data. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation and were within the control range for the different tester strains and positive control substances. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method).
Based on the results of Experiment 1, the same maximum dose level (5000 μg/plate) was employed in the second mutation test (pre-incubation method). The test item induced a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains, initially from 1500 μg/plate in the absence of metabolic activation (S9-mix). No toxicity was noted for Escherichia coli strain WP2uvrA dosed in either the absence or presence of S9-mix or any of the Salmonella strains dosed in the presence of S9-mix. A test item film (white and cloudy in appearance) was noted at 5000 μg/plate in both the presence and absence of metabolic activation (S9-mix) in Experiments 1 and 2. This observation did not prevent the scoring of revertant colonies.
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method).
Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). One statistically significant value was noted (TA100 at 50 μg/plate in the absence of metabolic activation (S9-mix). However, as the maximum fold increase was only 1.3 times the concurrent vehicle control and there was no evidence of a dose-related response or reproducibility, the response was considered an accidental finding reflecting biological variance and not being of toxicological relevance. Therefore, this value has not been highlighted in Table 4 as it did not meet the required criteria for a positive response.
In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test the test item was considered to be non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in bacteria

An Ames Test according to OECD Guideline 471 and GLP was performed. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 (plate incorporation) was based on OECD TG 471 and was 1.5 to 5000 μg/plate. The experiment was performed on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 5 to 5000 μg/plate. Seven test item concentrations were selected in Experiment 2 in order to ensure the study achieved at least four non-toxic dose levels as required by the test guideline, and were selected based on the lack of cytotoxicity noted in Experiment 1 and the potential for a change in the cytotoxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.
The vehicle (acetone) control plates gave counts of revertant colonies within the normal range of the historical control data. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation and were within the control range for the different tester strains and positive control substances. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method).
Based on the results of Experiment 1, the same maximum dose level (5000 μg/plate) was employed in the second mutation test (pre-incubation method). The test item induced a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains, initially from 1500 μg/plate in the absence of metabolic activation (S9-mix). No toxicity was noted for Escherichia coli strain WP2uvrA dosed in either the absence or presence of S9-mix or any of the Salmonella strains dosed in the presence of S9-mix. A test item film (white and cloudy in appearance) was noted at 5000 μg/plate in both the presence and absence of metabolic activation (S9-mix) in Experiments 1 and 2. This observation did not prevent the scoring of revertant colonies.
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method).
Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). One statistically significant value was noted (TA100 at 50 μg/plate in the absence of metabolic activation (S9-mix). However, as the maximum fold increase was only 1.3 times the concurrent vehicle control and there was no evidence of a dose-related response or reproducibility, the response was considered an accidental finding reflecting biological variance and not being of toxicological relevance. Therefore, this value has not been highlighted in Table 4 as it did not meet the required criteria for a positive response.
In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test the test item was considered to be non-mutagenic.

 

Gene mutation in mammalian cells

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.

 

Chromosome aberration in mammalian cells

The purpose of this study was the detection of structural chromosomal aberrations in cultured mammalian cells. The GLP-compliant study was performed according to OECD Guideline 473.
Duplicate cultures of human lymphocytes, treated with the test item, and quadruplicate cultures for the solvent controls, were evaluated for chromosome aberrations at up to four dose levels, together with solvent 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 Cell Growth Inhibition Test (Preliminary Toxicity Test) where the results indicated that the maximum concentration should be limited by the onset of test item precipitate in the 4-hour exposure groups in both the absence and presence of metabolic activation, and test item-induced toxicity in the 24-hour exposure group in the absence of metabolic activation. The dose levels selected for the Main Test were as follows:

4(20)-hour without S9
0, 5, 10, 20, 40, 60, 80, 120, 160 µg/mL
4(20)-hour with S9 (2%)
0, 5, 10, 20, 40, 60, 80, 120, 160 µg/mL
24-hour without S9
0, 5, 10, 20, 30, 40, 60, 80, 160 µg/mL

All solvent (acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.
All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level in the 4-hour exposure group in the absence of metabolic activation, and a dose range that achieved optimum levels of test item-induced toxicity in the 24-hour exposure group in the absence of metabolic activation.
In the presence of metabolic activation there was a small but statistically significant increase compared to the concurrent control in the frequency of cells with aberrations at 80 μg/mL (lowest precipitating dose level). The response at 80 μg/mL was only break type aberrations (1.7 % ctb), which was well within the laboratory historical control range (0 - 2 %) for a vehicle, the vehicle on this occasion was also very low, therefore it was considered to be an artifactual response which was within normal variation and of no toxicological relevance. There was no concentration related response demonstrated when evaluated with the trend test. The P value from the trend test was 0.162 and was therefore not statically significant.
As a conclusion, the test item, was considered to be non-clastogenic to human lymphocytes in vitro both in the presence and absence of a metabolic activation system (rat liver S9 mix).

Justification for classification or non-classification

Based on the results presented the registered substance is not subject to classification and labelling according to Regulation (EC) No 1272/2008.