N,N''-(methylenedi-4,1-phenylene)bis[N'-(4-methylphenyl)urea]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD TG 471): negative

In vitro chromosome aberration test (OECD TG 473): negative

Gene mutation in mammalian cells (OECD TG 490): negative

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:

26 November 1993 - 17 December 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with S9) TA100: 1.0, 3.3, 10.0, 33.3, 100, 333, 1000, 3330 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate
Experiment 2:
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle:
A homogeneous suspension could be in DMSO and DMSO is accepted and approved by authorities and international guidelines.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9, 5 µg/plate in saline for TA1535

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without S9, 60 µg/plate in saline for TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Daunomycine

Remarks:

Without S9, 4 µg/plate in saline for TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9, 650 µg/plate in DMSO for TA100

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9, 5 µg/plate in DMSO for TA1535, TA1537, 0.5 µg/plate in DMSO for TA98, TA100

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Rationale for test conditions:

Selection of an adequate range of doses was based on a preliminary toxicity test with strain TA100, both with and without 59-mix.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the Ames test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic)in the Ames test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant. If the test substance showed in the first test only a positive response at one or two concentrations, the assay was repeated with doses just below and exceeding those showing positive effects in the first test.
b) The positive response should be reproducible in at least one independently repeated experiment.

Statistics:

No formal hypothesis testing was done.

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

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

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

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Slight precipitation was observed at dose levels of 1000 and 3330 µg/plate, moderate precipitation was observed at 5000 µg/plate.

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

Conclusions:

In an AMES test, performed according to OECD guideline and in accordance with GLP principles, KY-AF was found not to be mutagenic with or without metabolic activation.

Executive summary:

An AMES test was performed according to OECD guideline 471 and in accordance with GLP principles. All bacterial strains showed negative responses up to 5000 ug/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. Precipitation of the test substance was observed at and above 1000 µg/plate. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that KY-AF is not mutagenic in the Salmonella typhimurium reverse mutation assay with or without metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 July 2017 - 12 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

- E. coli: Tryptophan gene

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with S9): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
Experiment 2:
Without and with S9-mix: 5.4, 17, 52, 164 and 512 μg/plate

Vehicle / solvent:

- Vehicle used: ethanol
- Justification for choice of vehicle: A solubility test was performed based on visual assessment. At concentrations of 0.054 mg/ml and higher KY-AF was suspended in ethanol. At a concentration of 0.017 mg/ml the test item was dissolved in ethanol. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension.

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

other: With S9, 2-aminoanthracene 15 μg/plate in DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Rationale for test conditions:

Selection of an adequate range of doses was based on a preliminary toxicity test, both with and without 59-mix.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a. The total number of revertants in tester strain WP2uvrA is not greater than two (2) times the concurrent control.
b. The negative response should be reproducible in at least one follow up experiment.
A test item is considered positive (mutagenic) in the test if:
a. The total number of revertants in tester strain WP2uvrA is greater than two (2) times the concurrent control.
b. In case a follow up experiment is performed when a positive response is observed, the positive response should be reproducible in at least one follow up experiment.

Statistics:

Not performed.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
First mutation experiment: Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 512 μg/plate and upwards and at 164 μg/plate and upwards at the end of the incubation period.
Second experiment: Precipitation of the test item on the plates was observed at the start and at the end of the incubation period at the concentrations of 164 and 512 μg/plate.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
S9-mix - +
Range 93 – 1951 93 - 1359
Mean 1128 422
SD 484 151
n 1679 1728
SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between May 2015 and May 2017.

- Negative (solvent/vehicle) historical control data:
S9-mix - +
Range 10 – 59 9 - 69
Mean 25 31
SD 7 8
n 1739 1745
SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between May 2015 and May 2017.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No reduction of the bacterial background lawn and no decrease in the number of revertants were observed.

Conclusions:

In an Escherichia coli reverse mutation assay, performed according to OECD 471 guideline and in accordance with GLP principles, KY-AF was found not to be mutagenic with or without metabolic activation.

Executive summary:

An Escherichia coli reverse mutation assay was performed according to OECD guideline 471 and in accordance with GLP principles.

The strain WP2 uvr A showed negative responses up to and including 5000 μg/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. Precipitation of the test substance was observed at and above 512 μg/plate. No reduction of the bacterial background lawn and no decrease in the number of revertants were observed.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that KY-AF is not mutagenic in the Escherichia coli reverse mutation assay with or without metabolic activation.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 November 1999 - 12 January 2000

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)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

Type and identity of media:
- Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Within 4 h after blood collection lymphocyte cultures were started.
- Culture medium
Culture medium consisted of Ham's F10 medium without thymidine and hypoxanthine (Gibco), supplemented with 20% (v/v) heatinactivated (56°C; 30 min) foetal calf serum (Gibco), L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 µg/ml respectively), sodium bicarbonate (1.2 g/L) and 30 U/ml heparin.
- Lymphocyte cultures
Whole blood was cultured in F10 complete culture medium with Phytohaemagglutinin (Murex). Per culture (in the absence of S9-mix 5 ml F10 complete culture medium and in the presence of S9-mix 4.8 ml F10 complete culture medium and 0.4 ml whole blood) 0.1 ml (9 mg/ml) Phytohaemagglutinin was added.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not applicable, immediately after blood collection lymphocyte cultures were started.
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: not applicable, immediately after blood collection lymphocyte cultures were started.

Average Generation Time (AGT) of the cells
dose range finding study : age 27, AGT = 15.5 h (Dec. 1999)
experiment 1: age 33, AGT = 16.4 h (Dec. 1999)
experiment 2: age 21, AGT = 15.5 (Dec. 1999)

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

Dose range finding test:
Without S9-mix, 3hr exposure; 24 hr fixation: 1, 3, 10, 33 and 100 µg/mL
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 1, 3, 10, 33 and 100 µg/mL
With S9-mix, 3hr exposure; 24 hr fixation: 1, 3, 10, 33 and 100 µg/mL
First cytogenetic test:
Without S9-mix, 3 h exposure time, 24 h fixation time: 10, 33 and 100 µg/mL
With S9-mix, 3 h exposure, 24 h fixation time: 10, 33 and 100 µg/ mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 10, 33 and 100 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 10, 33 and 100 µg/mL
With S9-mix, 3 hr exposure; 48 hr fixation: 10, 33 and 100 µg/mL

At a concentration of 100 µg/ml KY-AF precipitated in the culture medium. Therefore, a concentration of 100 µg/ml was used as the highest concentration of KY-AF.

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: A homogeneous suspension could be obtained in DMSO and DMSO is accepted and approved by authorities and international guidelines

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9; in Hank's Balanced Salt Solution: 0.5 µg/mL for a 3 h exposure period, 0.2 µg/mL for a 24 h exposure period and 0.1 µg/mL for a 48 h exposure period

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9; in Hank's Balanced Salt Solution: 15 µg/mL for a 3 h exposure periode

Details on test system and experimental conditions:

TEST SYSTEM
Cultured peripheral human lymphocytes

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Rationale for test conditions:

Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assay considering the highest dose level is limited by the solubility.

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
- It induced a dose-related statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
- A statistically significant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if:
- None of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included and excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Species / strain:

lymphocytes: Cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not specified
- Effects of osmolality: Not specified

- Precipitation: Precipitation in the exposure medium was observed at dose levels of 100 µg/ml

RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to and including the highest precipitating tested dose

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide induced appropriate responses.

Conclusions:

A chromosome aberration study with KY-AF was performed according to OECD guideline 473 and in accordance with GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that KY-AF is not clastogenic in human lymphocytes.

Executive summary:

In a chromosome aberration study, cultured peripheral human lymphocytes were exposed to different concentrations of KY-AF (dissolved in DMSO), in the presence and absence of S9-mix according to OECD/ EC guidelines and in accordance with GLP principles. KY-AF precipitated in the culture medium at a concentration of 100 µg/ml. In the first cytogenetic assay, KY-AF was tested up to precipitating concentration for a 3 h exposure time with a 24 h fixation time (100 μg/ml). In the second cytogenetic assay, KY-AF was tested up to and including 100 μg/ml for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix and for a 3 h treatment time with a 48 h fixation time in the presence of S9. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. KY-AF did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments. No effects of KY-AF on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that KY-AF is not clastogenic in the chromosome aberration test with human lymphocytes.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 July 2017 - 14 August 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)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Specific details on test material used for the study:

- The test item is irritant or corrosive.

Target gene:

Thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: L5178Y/TK+/- -3.7.2C mouse lymphoma cells from American Type Culture Collection, (ATCC, Manassas, USA), (2001)
- Suitability of cells: recommended test system in international guidelines

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
* Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin
* Growth medium: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
* Exposure medium: 3 hr exposure: basic medium, supplemented with 5% (v/v) heat-inactivated horse serum (=R5 medium); 24 hr exposure: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
* Selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 μg/ml trifluorothymidine
* Non-selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium)
- Properly maintained: yes, stock cultures of the cells were stored in liquid nitrogen (-196°C).
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).

Test concentrations with justification for top dose:

The test item precipitated in the exposure medium at concentrations of 7.8 μg/ml and above. The test item was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test item concentration for the dose-range finding test was 20 μg/mL.
- Dose-range finding test (with and without S9; cytotoxicity test): 1.25, 2.5, 5, 10 and 20 μg/mL
- Experiment 1 (with and without S9) and experiment 2 (without S9): 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10 and 20 μg/mL.

Vehicle / solvent:

- Vehicle used: ethanol
- Justification for choice of vehicle: as recommended in OECD test guideline 490

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): below 1 x 10^6 cells/mL

DURATION
- Exposure duration:
Short term treatment: 3 hours (with and without S9-mix)
Prolonged treatment: 24 hours (without S9-mix).
- Expression time: 2 days in which at least 4 x 10^6 cells were subcultured every day
- Selection time (if incubation with a selection agent): 11 or 12 days

SELECTION AGENT: 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- test concentrations: 1
- positive control: 1
- solvent control: 2

NUMBER OF CELLS EVALUATED: 9.6 x 10^5 cells per concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (see 'Any other information on materials and methods incl. tables' for details on calculations)

Evaluation criteria:

The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
- A test item is considered positive (mutagenic) in the mutation assay if it induces a mutation frequency of more than the mutation frequency in the controls + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
- A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
- A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of the mutation frequency in the controls + 126.

ACCEPTABILITY CRITERIA:
a) The absolute cloning efficiency of the solvent controls is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The suspension growth over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10^-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10^-6).

Key result

Species / strain:

mouse lymphoma L5178Y cells

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 applicable

Positive controls validity:

valid

Additional information on results:

DOSE-RANGE FINDING STUDY
- Both in the absence and presence of S9-mix, in the 3-hour exposure period, no toxicity in the relative suspension growth was observed up to a test item concentration of 20 μg/mL compared to the solvent control.
- In the 24-hour exposure period (without S9-mix), no toxicity in the relative suspension growth was observed up to a test item concentration of 20 μg/mL compared to the solvent control.
- The test item precipitated in the exposure medium at the highest concentration tested (20 μg/mL) in both exposure periods (3 hours and 24 hours)

FIRST AND SECOND EXPERIMENT (Mutation experiments)
The test item precipitated in the exposure medium at the test item concentration of 20 μg/mL.
No significant toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix in both experiments.
No significant increase in the mutation frequency at the TK locus was observed after treatment with KY-AF either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the KY-AF treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

HISTORICAL DATA: see table 1 and table 2

ACCEPTABILITY CRITERIA
- The positive controls both produced significant increased in the mutation frequency, which was within the 95% control limits of the historical data range.
- The absolute cloning efficiency of the solvent controls (CEday2) was 80 and 97 (without S9) and 99 and 93 (with S9) in experiment 1 and 85 and 93 in experiment 2.
- The spontaneous mutation frequency in the solvent control was 85 and 93 (without S9) and 99 and 93 (with S9) per 10^6 survivors in experiment 1 and 107 and 111 per 10^6 survivors in experiment 2. These frequencies were within the 95% control limits of the historical data range.
- The suspension growth over the two-day expression period for cultures treated with ethanol was between 20 and 23 (3 hour treatment) and 63 and 69 (24 hour treatment).

Table 1 Historical Control Data of the Spontaneous Mutation Frequencies of the Solvent Controls for the Mouse Lymphoma Assay

	Mutation frequency per 10^6survivors
	- S9-mix		+ S9-mix
	3 hour treatment	24 hour treatment	3 hour treatment
Mean	86	81	87
SD	23	26	28
n	220	202	273
Upper control limit (95% control limits)	135	135	145
Lower control limit (95% control limits)	37	28	28

SD = Standard deviation; n = Number of observations

Table 2 Historical Control Data of the Mutation Frequencies of the Positive Controls for the Mouse Lymphoma Assay

	Mutation frequency per 10^6survivors
	- S9-mix		+ S9-mix
	3 hour treatment	24 hour treatment	3 hour treatment
Mean	857	688	1710
SD	246	187	815
n	110	102	139
Upper control limit (95% control limits)	1425	1124	4214
Lower control limit (95% control limits)	289	253	-793

SD = Standard deviation; n = Number of observations

Distribution historical control data from experiments performed between January 2013 and November 2016. 

Conclusions:

KY-AF is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Executive summary:

A gene mutation test in mammalian cells was performed according to OECD guideline 490 and GLP principles, to assess the potential of KY-AF to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. The test item was tested up to and including a dose of 20 μg/mL was tested in the absence of S9 in a 3 and 24 hour treatment period and in the presence of S9 in a 3 hour treatment period. No toxicity was observed at this dose level in the absence and presence of S9-mix. KY-AF precipitated in the culture medium at this dose level.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. In both experiments, the test item did not induce an increase in the mutation frequency. Therefore, KY-AF is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test:

An AMES test was performed according to OECD guideline 471 and in accordance with GLP principles. All bacterial strains showed negative responses up to 5000 ug/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. Precipitation of the test substance was observed at and above 1000 µg/plate. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that KY-AF is not mutagenic in the Salmonella typhimurium reverse mutation assay with or without metabolic activation.

An Escherichia coli reverse mutation assay was performed according to OECD guideline 471 and in accordance with GLP principles.

The strain WP2 uvr A showed negative responses up to and including 5000 μg/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. Precipitation of the test substance was observed at and above 512 μg/plate. No reduction of the bacterial background lawn and no decrease in the number of revertants were observed.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that KY-AF is not mutagenic in the Escherichia coli reverse mutation assay with or without metabolic activation.

Chromosome aberration test:

In a chromosome aberration study, cultured peripheral human lymphocytes were exposed to different concentrations of KY-AF (dissolved in DMSO), in the presence and absence of S9-mix according to OECD/ EC guidelines and in accordance with GLP principles. KY-AF precipitated in the culture medium at a concentration of 100 µg/ml. In the first cytogenetic assay, KY-AF was tested up to precipitating concentration for a 3 h exposure time with a 24 h fixation time (100 μg/ml). In the second cytogenetic assay, KY-AF was tested up to and including 100 μg/ml for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix and for a 3 h treatment time with a 48 h fixation time in the presence of S9. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. KY-AF did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments. No effects of KY-AF on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that KY-AF is not clastogenic in the chromosome aberration test with human lymphocytes.

Mouse lymphoma test:

A gene mutation test in mammalian cells was performed according to OECD guideline 490 and GLP principles, to assess the potential of KY-AF to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. The test item was tested up to and including a dose of 20 μg/mL in the absence of S9 in a 3 and 24 hour treatment period and in the presence of S9 in a 3 hour treatment period. No toxicity was observed at this dose level in the absence and presence of S9-mix. KY-AF precipitated in the culture medium at this dose level.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. In both experiments, the test item did not induce an increase in the mutation frequency. Therefore, KY-AF is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Justification for classification or non-classification

Based on the results of the Ames test, in vitro chromosome aberration and mouse lymphoma studies, the substance KY-AF does not have to be classified for genotoxicity in accordance with Regulation (EC) No 1272/2008 and its amendments.