Reaction mass of Phosphorous(1+), (N-ethylethanaminato) diphenyl(phenylmethyl)-(T-4)-salt with 4, 4' - [2,2,2-trifluoro-1-(trifluoromethyl) ethylidene bis[phenol] (1:1) and (4,4'-[2,2,2-Trifluoro-1-(trifluoromethyl)ethylidene]diphenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genetic toxicity of the Reaction mass of AminoPhosphonium salt and BisphenolAF (XA 31)was assessed in 3 in vitro study conducted in compliance with the principles of Good Laboratory Practices:

- a gene mutation assay on Salmonella typhimurium TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA (OECD guideline 471).

- an in vitro chromosomal aberration assay in cultured human lymphocytes (OECD guideline 473).

- an in vitro mammalian cell gene mutation test using the thymidine kinase gene in Mouse Lymphoma cells (OECD guidelin 490, MLA).

In all three studies neagative results were reported in the presence and absence of metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 2013 - November 2013

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 37060B
- Expiration date of the lot/batch:
- Purity test date: >99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Plate Incorporation Method (Experiment 1, range finding test):
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.
The maximum concentration was 5000 µg/plate (the maximum recommended dose level).

Pre-Incubation Method (Experiment 2, main test)):
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 0.5 to 500 µg/plate for the Salmonella strains and 5 to 5000 µg/plate for Escherichia coli strain WP2uvrA.

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The test item was fully soluble in dimethyl sulphoxide at 50 mg/mL in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Plate Incorporation Method (experiment , range-finding test): in agar (plate incorporation);
- Pre-Incubation Method (experiment 2, main test): preincubatio

Plate Incorporation Method (experiment , range-finding test):
The test was performed with and without activation (S9-mix).
Each concentration of the test item, appropriate positive control, and each bacterial strain, was assayed using triplicate plates.
All of the plates were incubated at 37 °C± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were performed to confirm the automated scores.

Pre-Incubation Method (experiment 2, main test):
As Experiment 1 (the range-finding test) was deemed negative, Experiment 2 (main test) was performed using the pre-incubation method in the presence and absence of metabolic activation.
The test was performed with and without activation (S9-mix).
All testing for this experiment was performed in triplicate.
All of the plates were incubated at 37 °C± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Some manual counts were required due to the colonies spreading slightly.

Evaluation criteria:

1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al, 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated 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

Vehicle controls validity:

valid

Untreated 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

Vehicle controls validity:

valid

Untreated 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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The maximum dose level of the test item in Experiment 1 (plate incorporation method) was selected as the maximum recommended dose level of 5000 (µg/plate. There was a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 150 |ng/plate in the absence of metabolic activation (TA100) and at and above 500 µg/plate in the presence of metabolic activation (all Salmonella strains). Consequently in the second mutation test (pre-incubation method), the same maximum dose level was used for Escherichia colt strain WP2uvrA and the toxic limit (500 µg/plate) for the Salmonella strains. Once again the test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 50 µg/plate in the absence of metabolic activation (TA1537) and at and above 150 µg/plate in the presence of metabolic activation (TA1537 again). No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation.

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 in the range-finding test (plate incorporation method). Similarly, no significant 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 in the main test (pre-incubation method).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.

Conclusions:

Interpretation of results:
negative without metabolic activation
negative with metabolic activation

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 in the range-finding test (plate incorporation method). Similarly, no significant 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 in the main test (pre-incubation method).
Therefore, the test item was considered to be non-mutagenic under the conditions of this test.

Executive summary:

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008, 40 CFR 799.9510 TSCA bacterial reverse mutation test and the USA, EPA (TSCA) OCSPP harmonized guidelines.

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 pre­incubation 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 method) was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated 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 (plate incorporation method) and was 0.5 to 500 µg/plate for theSalmonellastrains and 5 to 5000 µg/plate for Escherichia coli strain WP2uvrA.

Additional dose levels and an expanded dose range were selected in Experiment 2 (pre-incubation method) in order to achieve both four non-toxic dose levels and the toxic limit of the test item.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in Experiment 1 (plate incorporation method) was selected as the maximum recommended dose level of 5000 µg/plate. There was a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 150 |ug/plate in the absence of metabolic activation (TA100) and at and above 500 µg/plate in the presence of metabolic activation (all Salmonella strains). Consequently in the second mutation test (pre-incubation method), the same maximum dose level was used for Escherichia coli strain WP2uvrA and the toxic limit (500 µg/plate) for the Salmonella strains. Once again the test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 50 µg/plate in the absence of metabolic activation (TA1537) and at and above 150 µg/plate in the presence of metabolic activation (TA1537 again). No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation.

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 in the range-finding test (plate incorporation method). Similarly, no significant 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 in the main test (pre-incubation method).

The Reaction mass of AminoPhosphonium salt and Bisphenol AF (XA 31) was considered to be non-mutagenic 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:

August 2013 - January 2014

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

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 37060B
- Expiration date of the lot/batch: 31 Decemeber 2018
- Purity test date: >99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: Cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source and Suitability of cells: from the peripheral circulation of a non-smoking volunteer who had been previously screened for suitability.
- Cell cycle length, doubling time or proliferation index: The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The mean value of the AGT for the pool of regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.
- cell and medium culuture:
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at approximately 37 °C with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.

Test concentrations with justification for top dose:

Preliminary Toxicity Test: The dose range of test item used was 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL.
Definitive test:
- Experiment 1: 1.88, 3.75, 7.5, 15, 30 and 60 ug/mL
- Experiment 2: 7.5, 15, 30, 60, 90 and 120 mL (4-hour exposure to the test item without S9-mix) and 1.88, 3.75, 7.5, 15, 30 and 60 ug/mL (%), followed by 20-hour culture in treatment)

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION:
- Preincubation period: 48 hours
- Exposure duration: 4hours (and 24 hours in the range test)
- Expression time (cells in growth medium):
- Fixation time (start of exposure up to fixation or harvest of cells): 20-hour

SELECTION AGENT (mutation assays):

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (Colcemid 0.1 ug/mL) two hours before the required harvest time.

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
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.
When the slides were dry they were stained in 5% slide for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

NUMBER OF CELLS EVALUATED:
cytotoxicity: 2000 lymphocyte

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index, A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported


- OTHER:
culture conditions: 9.05mL MEM, 10% (FBS), 0.1 mL Li-heparin, 0.1 mL phytohaemagglutinin 0and .75 mL heparinized whole blood

Evaluation criteria:

A test item can be classified as non-genotoxic if:
1. The number of induced chromosome aberrations in all evaluated dose groups is within the range of laboratory historical control data.
2. No toxicologically or statistically significant increase of the number of structural chromosome aberrations is observed following statistical analysis.
A test item can be classified as genotoxic if:
1. The number of induced structural chromosome aberrations is not in the range of laboratory historical control data.
And
2. Either a concentration-related or a statistically significant increase of the number of structural chromosome aberrations is observed. Marked increases only observed in one dose level will be assessed on a case by case basis

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.

Key result

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: see details below

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no


RANGE-FINDING STUDY: see table 1 in " Any other information on results including tables
Haemolysis was observed following exposure to the test item. In the 4(20)-hour exposure group (in the absence of S9), haemolysis was observed between 156.25 and 2500 µg/mL. In the 4(20)-hour exposure group in the presence of S9, haemolysis was observed at and above 156.25 µg/mL and at and above 78.13 |ug/mL in the 24-hour continuous exposure group. Haemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.
In the 4(20)-hour exposure groups, there were substantial decreases in mitotic index at the dose levels analyzed. In the 24-hour continuous exposure, increases in mitotic index were observed at both dose levels analyzed, possibly due to cell cycle delay.


HISTORICAL CONTROL DATA:
The number of cells with chromosome aberrations found in the vehicule and positive control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide induced appropriate responses (statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolizing system).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used : mitotic index
> experiment 1: In the absence of S9, 15%, 42% and 54% mitotic inhibition was achieved at 15, 30 and 60 µg/mL, respectively. In the presence of S9, a modest dose-related inhibition of mitotic index was observed at 30 and 60 µg/mL (15% and 37% mitotic inhibition, respectively). The maximum dose level selected for metaphase analysis was, therefore, 60 µg/mL for both exposure groups
> experiment 2: toxicity was apparent at and above 90 µg/mL in both the absence and presence of S9. However, in the 24-hour continuous exposure group, no dose-related inhibition of mitotic index was observed, and 42% mitotic inhibition was achieved at 15 µg/mL (the lowest dose level analyzed for chromosomal damage in this exposure group). In fact, the mitotic index increased with concentration. This phenomenon is indicative of cell cycle delay where the toxicity of the test item delays the cell cycle process causing synchronization of the cells, and confirmed the observed response in the preliminary toxicity test. In the presence of S9, inhibition of the mitotic index was observed but it was not clear cut with 66% and 50% mitotic inhibition occurring at 30 and 60 µg/mL, respectively (30 |ug/mL was considered too toxic to assess for metaphase analysis).
The maximum dose level selected for metaphase analysis was based on toxicity. The maximum dose levels selected for metaphase analysis were 45 |ug/mL and 60 µg/mL in the absence and presence of S9, respectively.

- polyploid:
> experiment 1: Although there was a small increase in polyploid cells at 60 µg/mL in the presence of S9, the increase was only just outside the historical maxima and was considered to have no biological relevance. Therefore, the test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.
> experiment 2: There was no incidence of polyploid cells at any dose level in either of the exposure groups. This confirms that the response observed in the presence of S9 in Experiment 1 was spurious and had no toxicological significance

CHROMOSOME ABBERATION:
> experiment 1: The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.
> experiment 2: The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

Table 1 : Mitotic Index - Preliminary Toxicity Test

Dose Level (Hg/mL)	4(20)-Hour Without S9		4(20)-Hour With S9		24-Hour Without S9
	Mitotic Index	%of Control	Mitotic Index	%of Control	Mitotic Index	%of Control
0	5.15	100	4.50	100	1.85	100
19.53	1.90	37	1.60	36	2.30	124
39.06	2.05	40	2.55	57	3.50	189
78.13	NM	-	NM	-	NMH	-
156.25	NMH	-	NMHP	-	NMH	-
312.5	NMHP	-	NMHP	-	NMHP	-
625	NMHP	-	NMHP	-	NMHP	-
1250	NMHP	-	NMHP	-	NMHP	-
2500	NMHP	-	NMHP	-	NMHP	-
5000	NMP	-	NMHP	-	NMHP	-

Validity criteria:

- All vehicle (dimethyl sulphoxide) 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 the satisfactory performance of the test and of the activity of the metabolizing system.

Conclusions:

Under these experimental conditions, no statistically significant increase in the frequency of cells with chromosome aberrations was observed, both with and without S9 mix, using a dose range from 1;88 to 5000 µg/ml). XA 31 did not show any clastogenic activity in the in vitro mammalian chromosome aberration test with cultured human lympocytes.

Executive summary:

The potential of the Reaction mass of AminoPhosphonium salt and Bisphenol AF (XA 31) to induce chromosome aberrations in cultured human lymphocytes was assessed according to the OECD guideline 473 and the EU Method B.10. The study was conducted in compliance with the principles of Good Laboratory Practice.

Duplicate cultures of human lymphocytes, exposed to the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study. In the Experiment 1, cultured human lympocytes were exposed to 1.88, 3.75, 7.5, 15, 30, 60 µg/mL of XA 31 for 4-hours 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 and for 4-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4-hour exposure with addition of S9 was repeated using a 1% final S9 concentration and dose levels of 3.75, 7.5, 15, 30, 45, 60, 90 µg/mL, whilst in the absence of metabolic activation the exposure time was increased to 24 hours and the dose levels were 7.5, 15, 30, 60, 90, 120  µg/mL .

The frequency of cells with structural chromosome aberrations of the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

XA 31 did not exhibit significant cytotoxicity and did not induce any statistically significant increases in the frequency of cells with aberrations in either of the two separate experiments for all tested concentrations.

Under these test conditions, the Reaction mass of AminoPhosphonium salt and Bisphenol AF (XA 31) did not show any clastogenic activity in the in vitro mammalian chromosome aberration test with cultured human lympocytes.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 2016 - May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

yes

Remarks:

considered not to have compromised the validity or integrity of the study

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

considered not to have compromised the validity or integrity of the study

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 37060B
- Expiration date of the lot/batch: March 2019.
- Purity test date: >99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

Target gene:

thymidine kinase, TK +/-, locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: TK+/- 3.7.2C subline of L5178Y cells were obtained from ATCC (American Type Culture Collection, Manassas, USA), through Biovalley (77601, Marne-La-Vallée, France).
- Suitability of cells:
- Cell cycle length, doubling time or proliferation index:
- Sex, age and number of blood donors if applicable:
- Whether whole blood or separated lymphocytes were used if applicable:
- Number of passages if applicable:
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes:
- Normal (negative control) cell cycle time:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
- Properly maintained: [yes/no]
- Periodically checked for Mycoplasma contamination: [yes/no]
- Periodically checked for karyotype stability: [yes/no)
- Periodically 'cleansed' against high spontaneous background: [yes/no]

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Experiments without S9 mix
The selected dose-levels were as follows:
- 0.63, 1.25, 2.5, 5, 10 and 20 µg/mL for the 3-hour treatment (First experiment)
- 1, 2, 4, 6, 10, 12, 14 and 28 µg/mL for the 24-hour treatment (Second experiment)

Experiment with S9 mix
The selected dose-levels were as follow:
- 0.63, 1.25, 2.5, 5, 10 and 20 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: According to available solubility data

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- in medium. i.e. the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% (3-hour treatment) or 10% (24-hour treatment) in a 37°C, 5% CO2 humidified incubator.
- Cell density: Cultures of 20 mL at 5 x 105 cells/mL (3-hour treatments) or cultures of 50 mL at 2 x 105 cells/mL (24-hour treatment)


DURATION
- Exposure duration: 3 or 24 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11-12 days with trifluorothymidine (TFT)

SELECTION AGENT (mutation assays): trifluorothymidine (TFT) at 4 μg/mL

NUMBER OF REPLICATIONS: Two independent experiments with two cultures per dose level (six dose-levels of the test item)

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression period (CE2).

DETERMINATION OF MUTAGENICITY/
- Method: The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.
- Differentiation of small and large colonies:
- size of small colonies: < 25% of the diameter of the well,
- size of large colonies: > 25% of the diameter of the well.

Evaluation criteria:

Evaluation of a positive response:
Based on IWGT recommendations, a test item is considered clearly positive if, in any of the experimental conditions examined:
- at least at one dose-level the mutation frequency minus the mutation frequency of the vehicle control (IMF) equals or exceeds the Global Evaluation Factor (GEF) of 126 x 10E-6,
- a dose-response relationship is demonstrated by a statistically significant trend test.

Evaluation of a negative response:
A test item is considered clearly negative if, in all experimental conditions, no dose-response relationship is demonstrated or, if there is an increase in MF, it does not exceed the GEF.

Noteworthy increases in the mutation frequency observed only at high-levels of cytotoxicity (Adj. RTG lower than 10%), but with no evidence of mutagenicity at dose-levels with Adj. RTG between 10 and 20%, are not considered as positive results.

A test item may be considered as non-mutagenic when there is no culture showing an Adj. RTG value between 10 and 20% if :
- there is at least one negative data point between 20 and 25% Adj. RTG and no evidence of mutagenicity in a series of data points between 100 and 20% Adj. RTG,
-there is no evidence of mutagenicity in a series of data points between 100 and 25% and there is also a negative data point between 10 and 1% Adj. RTG

Statistics:

To assess the dose-response relationship, a linear regression was performed between dose-levels and individual mutation frequencies obtained at dose-levels showing a mean Adj. RTG ≥ 10%. This statistical analysis was performed using SAS Enterprise Guide software.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see details in "additional information on results

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: At the highest tested dose-level (i.e. 125 µg/mL), the pH of the culture medium was approximately 7.4 (as for the vehicle control) and the osmolality was 435 mOsm/kg H2O (436 mOsm/kg H2O for the vehicle control). Thus, none of the selected dose-levels was considered to produce extreme culture conditions.
- Precipitation: A precipitate was observed in the culture medium at dose-levels ≥ 20 µg/mL at the end of the treatment periods.

RANGE-FINDING/SCREENING STUDIES:
Based on available solubility data, the dose-level of 125 µg/mL, obtained using a test item concentration of 25 mg/mL and a treatment volume of 0.5% (v/v) in the culture medium (i.e. 100 µL/20 mL culture medium for the 3-hour treatments and 250 µL/50 mL culture medium for the 24-hour treatment), was expected to produce precipitation in the culture medium. This dose-level was therefore selected as the top-dose for the preliminary test and the selected range was 0.25, 2.5, 12.5, 25, 62.5 and 125 µg/mL. A precipitate was observed in the culture medium at dose-levels ≥ 12.5 µg/mL at the end of the 3-hour treatment and at dose-levels ≥ 25 µg/mL at the end of the 24-hour treatment.

HISTORICAL CONTROL DATA
- Positive historical control data:For the positive control cultures, the increase in the mutation frequencies met also the acceptance criteria. In addition, the upper limit of cytotoxicity observed in the positive control cultures had an Adj. RTG greater than 10%.
- Negative (solvent/vehicle) historical control data: With one exception which was not considered to have compromised the integrity or validity of the study, the cloning efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria.

The study was therefore considered to be valid.

INFORMATION ON CYTOTOXICITY:
1) Preliminary cytotoxicity test:
Following the 3-hour treatment without S9 mix, a moderate to severe cytotoxicity was induced at dose levels ≥ 25 µg/mL, as shown by a 64 to 100% decrease in Adj. RTG.
Following the 24-hour treatment without S9 mix, a severe cytotoxicity was induced at dose-levels ≥ 12.5 µg/mL, as shown by a 94 to 100% decrease in Adj. RTG.
Following the 3-hour treatment with S9 mix, a marked to severe cytotoxicity was induced at dose-levels ≥ 62.5 µg/mL, as shown by a 85 to 100% decrease in Adj. RTG.

2) Main tests:
- Experiments without S9 mix:
No cytotoxicity was observed at any of the tested dose-levels following the 3-hour treatment, as shown by the absence of any noteworthy decrease in Adj. RTG.
Following the 24-hour treatment, a slight to severe cytotoxicity was induced at dose-levels ≥ 4 µg/mL, as shown by a 42 to 100% decrease in Adj. RTG.

- Experiment with S9 mix:
No cytotoxicity was observed at any of the tested dose-levels, as shown by the absence of any noteworthy decrease in Adj. RTG.


INFORMATION ON CYTOTOXICITY:
- Experiments without S9 mix:
Following the 3-hour treatment, no noteworthy increase in the mutation frequency was noted at any of the tested dose-levels, relative to the corresponding vehicle control, and no dose-response relationship was demonstrated by the linear regression.
Following the 24-hour treatment (Table 5), no noteworthy increase in the mutation frequency was noted at any of the tested dose-levels, relative to the corresponding vehicle control, and no dose-related increase was demonstrated by the linear regression. None of the tested dose-levels induced an Adj. RTG between 10 and 20% under these experimental conditions. However, there was one negative data point at 14 µg/mL, which induced an Adj. RTG of 24%, and there was no evidence of mutagenicity at the dose-levels ranging from 1 to 12 µg/mL, which induced Adj. RTGs between 25 and 64%.


- Experiment with S9 mix:
No noteworthy increase in the mutation frequency was noted at any of the tested dose-levels, relative to the corresponding vehicle control, and no dose-response relationship was demonstrated by the linear regression.
These results met the criteria of a negative response.

Conclusions:

Under the experimental conditions of this study, the test item did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.

Executive summary:

This study was performed to investigate the potential of the test item XA31 to induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK^+/-mouse lymphoma cells. The study was performed according to OECD guideline no. 490 and EC guideline n° B17 and in compliance with the Principles of Good Laboratory Practice.

 

The test item XA31, dissolved in dimethylsulfoxide (DMSO), was tested in two independent experiments, with or without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Cultures of mouse lymphoma cells were exposed 3-hours or 24-hours to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% (3-hour treatment) or 10% (24-hour treatment) in a, 5% CO₂humidified incubator. For the 24-hour treatment, flasks were gently shaken at least once.

Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE₂). The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.

Since the test item was found cytotoxic and poorly soluble in the culture medium, the selection of the highest dose-level for the main experiments was based both on the level of precipitate and on the level of cytotoxicity (i.e.decrease in Adj. RTG).

In the experiments without S9 mix,the selected dose-levels were 0.63, 1.25, 2.5, 5, 10 and 20 µg/mL for the 3-hour treatment and 1, 2, 4, 6, 10, 12, 14 and 28 µg/mL for the 24-hour treatment.

In the experiments with S9 mix, the selected dose-levels were 0.63, 1.25, 2.5, 5, 10 and 20 µg/mL (3-hour treatment only).

 

Following the 3-hour treatment either with or without S9 mix as well as the 24-hour treatment without S9 mix, no noteworthy increase in the mutation frequency was noted at any of the tested dose-levels, relative to the corresponding vehicle control, and no dose-response relationship was demonstrated by the linear regression.

With one exception which was considered not to have compromised the integrity or validity of the study, the cloning efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria. For the positive control cultures, the increase in the mutation frequencies met also the acceptance criteria

 

In conclusion, the test item XA31 did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

GENETIC TOXICIY IN VITRO:

1. Gene mutation in bacteria:

The mutagenic activity of the Reaction mass of AminoPhosphonium salt and BisphenolAF was assessed in the Ames test performed in five strains of Salmonella typhimurium TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA according to the OECD guideline 471 and the EU Method B13/14. The study was in compliance with the Principles of Good Laboratory Practice. Reaction mass of AminoPhosphonium salt and BisphenolAF was tested 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 method) was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated 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 (plate incorporation method) and was 0.5 to 500 µg/plate for the Salmonella strains and 5 to 5000 µg/plate for Escherichia coli strain WP2uvrA.

 

The numbers of revertants for the vehicle and positive controls were as specified in the acceptance criteria. The study was therefore considered valid. Reaction mass of AminoPhosphonium salt and BisphenolAF did not induce 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 in the range-finding test (plate incorporation method). Similarly, no significant 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 in the main test (pre-incubation method).

 

Reaction mass of AminoPhosphonium salt and BisphenolAF (XA 31) was considered to be non-mutagenic under the conditions of this test.