5-Brom-2-[(3,4,5-trifluorphenoxy)-difluormethyl]-1,3-difluorbenzene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471, RL1): negative with and without metabolic activation

In vitro HPRT test (OECD 476, RL1): negative with and without metabolic activation

In vitro chromosomal aberration test (OECD 473, RL1): negative with and without metabolic activation

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Oct 2017 - 31 Jan 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Blood samples were drawn from healthy non-smoking donors not receiving medication. For this study, blood was collected from a male donor (23 years old) for Experiment I and from a male donor (34 years old) for Experiment II. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of chromosomal aberrations in their peripheral blood lymphocytes.
Human lymphocytes were stimulated for proliferation by the addition of the mitogen phytohemagglutinin to the culture medium for a period of 48 hours. The cell harvest time point was approximately 1.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (Phenobarbital/β-naphthoflavone induced rat liver S9, mixed with S9 cofactor solution)

Test concentrations with justification for top dose:

Experiment I: Exposure period 4 h
with S9-mix: 4.4, 7.7, 13.5, 23.7, 41.5, 72.6, 127, 222, 667, 2000 µg/mL
without S9-mix: 4.4, 7.7, 13.5, 23.7, 41.5, 72.6, 127, 222, 667, 2000 µg/mL
Experiment II: Exposure period 22 h
without S9-mix: 6.4, 12.7, 16.6, 21.5, 28.0, 36.4, 47.3, 61.5, 80.0, 120 µg/mL

Vehicle / solvent:

DMSO (0.5 % v/v)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Culture conditions
Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

Pre-experiment
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterized by the percentages of mitotic suppression in comparison to the controls by counting 1000 cells per culture in duplicate. The experimental conditions in this pre-test phase were identical to those required and described below for the main experiment.
The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and solvent and positive controls. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 22 hrs after start of the exposure.

Cytogenetic Experiment
Pulse exposure
About 48 hrs after seeding, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with serum-free medium containing the test item. For the treatment with metabolic activation, 50 μL S9 mix per mL culture medium was added. After 4 hrs the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were resuspended in and washed with "saline G" (pH 7.2, containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose •H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4). The washing procedure was repeated once as described. After washing, the cells were resuspended in complete culture medium (with 10 % FBS) and cultured until preparation of the cells.
Continuous exposure (without S9 mix)
About 48 hrs after seeding, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with complete medium (with 10 % FBS) containing the test item. The culture medium was not changed until preparation of the cells.
Preparation of metaphases
Cultures were treated with the metaphase-arresting substance colcemid (final concentration: 0.2 μg/mL) approximately three hours before the requested harvest time. The cultures were harvested by centrifugation 22 hrs after beginning of treatment. The supernatant was discarded and the cells were resuspended in hypotonic solution (0.0375 M KCl). Then the cell suspension was allowed to stand at 37 °C for 20 minutes. After removal of the hypotonic solution by centrifugation (approx. 900 x g), the cells were fixed with a mixture of methanol and glacial acetic acid (3+1 parts, respectively). A small amount of cell suspension was then dropped onto clean, wet microscope slides and allowed to dry. The slides were stained with Giemsa, and, after drying covered with a cover slip. All slides were labelled with a computer-generated random code to prevent scorer bias.

2000 μg/mL were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 4.4 to 2000 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, precipitation of the test item was observed at the end of treatment at 41.5 μg/mL and above. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
Clear toxic effects were observed after 4 hours treatment in the absence of S9 mix at 72.6 μg/mL, only. 120 μg/mL were chosen as top treatment concentration for Experiment II.

Evaluation of cytotoxicity and cytogenetic damage
Evaluation of the slides was performed according to the standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik" using microscopes with 100 x oil immersion objectives.
Cytotoxicity is characterized by the percentages of mitotic suppression in comparison with the controls by counting 1000 cells per culture in duplicate.
At least 150 well-spread metaphases were evaluated per culture for structural aberrations. Only metaphases containing a number of centromeres equal to a number of 46 ± 2 were included in the analysis. Breaks, fragments, deletions, exchanges and chromosomal disintegrations are recorded as structural chromosomal aberrations. Gaps were recorded as well, but they are not included in the calculation of the aberration rates since gaps are achromatic lesions of unknown biological relevance for which a clear relationship to treatment cannot be established.

Rationale for test conditions:

Acceptability Criteria
The chromosomal aberration assay is considered acceptable if it meets the following criteria (OECD guideline 473):
a) The number of aberrations found in the solvent controls falls within the 95% control limits of the distribution of the laboratory’s historical negative control database. If they fall outside those limits, they are acceptable as long as these data are not extreme outliers and there is evidence that the test system is ‘under control’ and technical or human failure can be excluded.
b) The rate of chromosomal aberrations in the positive controls was statistically significantly increased compared with the concurrent negative control and compatible with those generated in the historical positive control data base.
c) Cell proliferation criteria in the solvent control were fulfilled by ensuring sufficient number of cells has reached mitosis and cytotoxicity levels were acceptable. For primary lymphocyte cultures the mitotic index (MI) is an appropriate measure of cytotoxicity.
d) The test item is tested with and without metabolic activation for 4 h, and sampled to a time equivalent to about 1.5 normal cell cycle lengths after the beginning of treatment, as well as a long treatment experiment without metabolic activation was performed. For the long treatment the cells were exposed to the test item continuously until sampling for a time equivalent to 1.5 normal cell cycle lengths. All three experimental conditions will be tested unless one part turns out positive.
e) At least three test concentrations that meet the acceptability criteria were evaluated and at least 300 well-spread metaphases were counted.

Evaluation criteria:

Interpretation of Results
A test substance is classified as non-clastogenic if:
a. none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b. there is no concentration-related increase when evaluated with an appropriate trend test,
c. all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits)

A test substance is classified as clastogenic if all of the following criteria are met:
a. at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b. the increase is dose-related when evaluated with an appropriate trend test,
c. any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits)
A test substance is classified as clastogenic if all of the following criteria are met:
a. at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b. the increase is dose-related when evaluated with an appropriate trend test,
c. any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits)

Key result

Species / strain:

lymphocytes: human

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: no influence
- Data on osmolality: no influence
- Precipitation and time of the determination: Experiment I (+/- S9 mix) at 41.5 µg/mL and above; Experiment II (-S9 mix) at 120 µg/mL and above; precipitation occured at the end of the experiment
- Definition of acceptable cells for analysis: At least 150 metaphases per culture were evaluated for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index

RANGE-FINDING/SCREENING STUDIES (if applicable):
2000 μg/mL were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 4.4 to 2000 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, precipitation of the test item was observed at the end of treatment at 41.5 μg/mL and above. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
Clear toxic effects were observed after 4 hours treatment in the absence of S9 mix at 72.6 μg/mL, only. 120 μg/mL were chosen as top treatment concentration for Experiment II.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
Vehicle control: DMSO
Positive control: EMS (- S9 mix), CPA (+ S9 mix)

For all test methods and criteria for data analysis and interpretation:
- Statistical analysis: Statistical analysis is confirmed by Fisher's exact test (modified) (p < 0.05)

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For lymphocytres in primary cultures: mitotic index (MI)
- Genotoxicity results (for both cell lines and lymphocytes)
o Definition for chromosome aberrations, including gaps : Breaks, fragments, deletions, exchanges and chromosomal disintegrations are recorded as structural chromosomal aberrations. Gaps were recorded as well, but they are not included in the calculation of the aberration rates since gaps are achromatic lesions of unknown biological relevance for which a clear relationship to treatment cannot be established.
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 excludling gaps : At least 150 well-spread metaphases were evaluated per culture for structural aberrations. Please refer to the attached backgroudn material for the number of cells with chromosomal aberrations
o Changes in ploidy (polyploidy cells and cells with endoreduplicated chromosomes): No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

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: Please refer to the attached background material.
- Negative (solvent/vehicle) historical control data: Please refer to the attached background material.

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro.
Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test, when tested up to moderate cytotoxic or precipitating concentrations.

Executive summary:

The test item, dissolved in DMSO, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments.

In each experimental group two parallel cultures were analyzed.Per culture at least 150 metaphases were evaluated for structural chromosomal aberrations.

The highest applied concentration in this study (2000 μg/mL of the test item) was chosen with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 473

In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed precipitation. In Experiment II in absence of S9 mix after continuous treatment, moderate cytotoxicity was observed at the highest evaluated concentration. The next higher tested concentrations, which were separated by a smaller factor than requested by the guideline, showed clear cytotoxicity exceeding the requested range. 

Either with or without metabolic activation neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro.

Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test, when tested up to moderate cytotoxic or precipitating concentrations.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Sep - 21 Nov 2017

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)

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:

hypoxanthine-guanine phosphoribosyl transferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

rat S9 mix

Test concentrations with justification for top dose:

Experiment I (- S9-mix): 3.9, 7.8, 15.6, 31.3, 62.5, 125.0, 250.0, 500.0 µg/mL
Experiment I (+ S9-mix): 7.8, 15.6, 31.3, 62.5, 125.0, 150.0, 500.0, 1000.0 µg/mL
Experiment IA (- S9-mix): 1.1, 2.2, 4.4, 8.8, 13.2, 17.5, 26.3, 35.0, 70.0 µg/mL

Vehicle / solvent:

DMSO, purity 99.99 %

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 6-8 days
SELECTION AGENT (mutation assays): 6-TG, 11 μg/mL
NUMBER OF EXPERIMENTS: 2 (- S9-mix), 1 (+ S9-mix)

Evaluation criteria:

Acceptability of the Assay
The gene mutation assay is considered acceptable if it meets the following criteria:
a) the mean values of the numbers of mutant colonies per 106 cells found in the solvent controls of both parallel cultures remain within the 95% confidence interval of the laboratory historical control data range.
b) the positive control substances should produce a significant increase in mutant colony frequencies and remain within the historical control range of positive controls.
c) the cloning efficiency II (absolute value) of the solvent controls must exceed 50 %.
The data of this study comply with the above mentioned criteria.

Evaluation of Results
A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
A mutagenic response is described as follows :
The test item is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits). The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares).

Statistics:

A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
A t-Test was performed using a validated test script of "R", a language and environment for statistical computing and graphics, to evaluate an isolated increase of the mutation frequency at a test point exceeding the 95% confidence interval. Again a t-test is judged as significant if the p-value (probability value) is below 0.05.
However, both, biological and statistical significance were considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The maximum test item concentration of the pre-experiment (2000 μg/mL) was chosen with the respect to the current OECD guideline 476 (2016). The maximum concentration of experiment 1 was limited by precipitation.
In experiment 1, strong cytotoxic effects occurred in both cultures in the absence of metabolic activation. Since the relative adjusted cloning efficiency 1 was far below 10% already at lower concentrations, this part was repeated (Experiment IA) using lower concentrations.
No relevant and reproducible increase in mutant colony numbers/10exp+6 cells was observed in the main experiments up to the maximum concentration.
Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, it is considered to be non-mutagenic in this HPRT assay.

Conclusions:

Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, it is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The maximum test item concentration of the pre-experiment (2000μg/mL) was chosen with the respect to the current OECD guideline 476 (2016). The maximum concentration of experiment 1 was limited by precipitation.

In experiment 1, strong cytotoxic effects occurred in both cultures in the absence of metabolic activation. Since the relative adjusted cloning efficiency 1 was far below 10% already at lower concentrations, this part was repeated (Experiment IA) using lower concentrations.

No relevant and reproducible increase in mutant colony numbers/10E+6 cells was observed in the main experiments up to the maximum concentration.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

 

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Jul 2006 - 13 Feb 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

HIS operon (S. thyphimurium)
TRP operon (E. coli)

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

his G 46, uvrB, rfa

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

his C 3076, uvrB, rfa

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

his D 3052, uvrB, rfa + R-factor

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

his G 46, uvrB, rfa + R-factor

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

his G 428, rfa + R-factor

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

E. coli WP2

Details on mammalian cell type (if applicable):

his C 3076, uvrB, rfa

Additional strain / cell type characteristics:

other: mutations in the tryptophan operon

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate (S9 mix) with standard co-factors with metabolic activation (Aroclor)

Test concentrations with justification for top dose:

The test material concentrations were used selected according to the EC and OECD guidelines for this test system :
1. Series: 5, 15.8, 50, 158, 500, 1580, and 5000 µg/plate
2. Series: 50, 158, 500, 1580 and 5000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

cumene hydroperoxide

other: Daunomycin, 2-Aminoanthracene

Details on test system and experimental conditions:

3 parallel plates were used for each concentration step pf the test material and the positive controls. Twice as many solvent control plates (here: DMSO) were used for each bacterial strain. The test was conducted in accordance with the plate incorporation methos described by Ames et al., 1975. Incubation of plates was performed at 37 °C for 2 to 3 days. Revertant coloniew were either scored using an Artek MiniCount colony counter or manually. The presence of background lawn was checked for each plate.

The assessment of test material-induced effects is dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test material concentration which shows the highest number of colonies.
The following criteria, based upon the historical controls of the laboratory and statistical considerations, are established:
-----------------------------------------------------------------------------------------
Mean Number of Colonies Maximal Mean Number of Colonies over the Actual
(Solvent Control) Solvent Control
(Test Material)
-----------------------------------------------------------------------------------------

<=10 <=9 >=30
<=30 <=19 >=40
<=80 <=29 >=80
<=200 <=49 >=120
<=500 <=79 >=200
Assessment No increase Clear increase

-----------------------------------------------------------------------------------------
All further results, ranging between "no" and "clear", are assessed as "weak increases".
Interpretations:
A test material is defined as non-mutagenic in this assay if "no" or "weak increases" occur in the first and second series of the main experiment.
("Weak increases" randomly occur due to experimental variation.)
A test material is defined as mutagenic in this assay if:
- a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;
- "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.
In all further cases, a third test series with the bacterial strain in question should be performed.
If the criteria for a positive test result are not fulfilled in at least two out of the three series, the test material is defined as being non-mutagenic in this test system.

Evaluation criteria:

Accepatance of positive and negative controls: The following mean numbers of revertants are acceptable as negative controls:
TA98: 15-60
TA100: 75-200
TA102: 200-450
TA1535: 3-37
TA1537: 4-31
WP2uvrA: 10-70

Statistics:

n.a.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Precipitation on agar plates: yes, at concentrations >/= 1580 µg/plate
Cytotoxicity: no

Conclusions:

With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

Executive summary:

Purpose

The purpose of this assay was to provide information on possible health hazards for the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in man.

Study Design

The investigations for the mutagenic potential of the test material were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed.

Results

The test material was dissolved in DMSO and tested at concentrations ranging from 5 to 5000 µg/plate. Precipitation of the test material on the agar plates was not at concentrations >/= 1580 µg/plate. No evidence for toxicity to the bacteria has been obtained.

Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9 -aminoacridine and cumene hydroperoxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test material showed no increase in the number of revertants of any bacterial strain. According to the criteria for negative and positive results predetermined in the Study Protocol (cf also "Criteria for negative and positive results"), the test material was not mutagenic under the described experimental conditions.

Conclusion

With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial Reverse Mutation Assay (OECD 471)

The investigations for the mutagenic potential of the test material were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed.

In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test material showed no increase in the number of revertants of any bacterial strain.

With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

 

In vitro Gene Mutation Assay in Mammalian Cells (OECD 476)

The study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The maximum test item concentration of the pre-experiment (2000μg/mL) was chosen with the respect to the current OECD guideline 476 (2016). The maximum concentration of experiment 1 was limited by precipitation.

In experiment 1, strong cytotoxic effects occurred in both cultures in the absence of metabolic activation. Since the relative adjusted cloning efficiency 1 was far below 10% already at lower concentrations, this part was repeated (Experiment IA) using lower concentrations.

No relevant and reproducible increase in mutant colony numbers/10E+6 cells was observed in the main experiments up to the maximum concentration. 

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

 

In vitro Cytogenicity Assay in Mammalian Cells (OECD 473)

The test item, dissolved in DMSO, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments.

The highest applied concentration in this study (2000 μg/mL of the test item) was chosen with respect to the current OECD Guideline 473.

In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed precipitation. In Experiment II in absence of S9 mix after continuous treatment, moderate cytotoxicity was observed at the highest evaluated concentration. The next higher tested concentrations, which were separated by a smaller factor than requested by the guideline, showed clear cytotoxicity exceeding the requested range. 

Either with or without metabolic activation neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro.

 

Justification for classification or non-classification

Based on the available data, there is no indication for a mutagenic or clastogenic potential in vitro and in vivo. The available data on genetic toxicity are conclusive but not sufficient to meet the criteria for classification according to Regulation (EC) 1272/2008.