Aluminum magnesium oxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECDTG 471): negative

In vitro chromosome aberration test (OECDTG 473): negative

Genemutation in mammalian cells (OECDTG 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:

30 July 2018 - 27 August 2018

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:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced with Aroclor 1254.

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 52, 164, 512, 1600, 2500 and 5000 μg/plate
Experiment 2, all five strains:
Without and with S9-mix: 5, 10, 100, 1000, 2500 and 5000 μg/plate
Experiment 3, TA100:
Without and with S9-mix: 100, 1000, 2500, 4000 and 5000 μg/plate

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: A solubility test was performed based on visual assessment. The test item formed a white homogenous suspension in DMSO. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. A correction factor of 1.13 for the purity/composition of the test item was applied in this study. Test item concentrations were used within 4 hours after preparation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191, 2.5 μg in DMSO for TA1537 (Direct plate assay)

Remarks:

Without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA) in DMSO, 1 μg for TA98 and TA100 (Direct plate assay), 2.5 μg for TA1535 and TA1537, 5 μg for TA100 (Pre-incubation assay), 15 μg for WP2uvrA

Remarks:

With S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 hours

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.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three 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 TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

Not performed.

Key result

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 applicable

Positive controls validity:

valid

Key result

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 applicable

Positive controls validity:

valid

Key result

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 applicable

Positive controls validity:

valid

Key result

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 applicable

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:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Experiment 1: Precipitation of KW-2200 on the plates was observed at the start of the incubation period at concentrations of 512 μg/plate and upwards and no precipitate was observed at the end of the incubation period.
Experiment 2: Precipitation of KW-2200 on the plates was observed at the start of the incubation period at the concentrations of 1000 and/or 2500 μg/plate and upwards and no precipitate was observed at the end of the incubation period.
Experiment 3: Precipitation of KW-2200 on the plates was observed at the start of the incubation period at the concentrations of 2500 μg/plate and upwards and no precipitate was observed at the end of the incubation period.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
TA1535 TA1537 TA98
S9-mix - + - + - +
Range 128 – 1530 73 – 1206 58 – 1407 54 – 1051 365 – 1978 250 – 1977
Mean 901 239 817 340 1355 903
SD 174 115 354 160 230 357
n 2400 2296 2051 2337 2357 2367

TA100 WP2uvrA
S9-mix - + - +
Range 439 – 1993 408 - 2379 93 – 1958 111 - 1359
Mean 905 1249 1059 444
SD 163 371 506 144
n 2402 2354 2153 2232

SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between May 2016 and May 2018.

- Negative (solvent/vehicle) historical control data:
TA1535 TA1537 TA98 TA100 WP2uvrA
S9-mix - + - + - + - + - +
Range 3 – 29 3 – 27 3 – 20 3 – 23 8 - 41 8 – 55 61 – 176 60 - 160 10 – 59 9 - 67
Mean 10 11 6 6 16 22 110 106 26 33
SD 3 4 2 3 5 7 17 20 6 8
n 2458 2426 2402 2352 2416 2458 2473 2398 2237 2217

SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between May 2016 and May 2018.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1: Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in the tester strains TA1535, TA1537 and TA98 in the absence and presence of S9-mix, except in tester strain TA1535 in presence of S9-mix.
Experiment 2: Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction of the bacterial background lawn, was observed in tester strain TA1535, TA1537, TA98 and TA100 in the absence of S9-mix and in tester strain TA98 in the presence of S9-mix.
Experiment 3: Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in the absence of S9-mix.

MUTAGENICITY:
In experiment 1 in tester strain TA100, the test item induced up to 2.3- and 2.2-fold increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively. The increases were more than two-fold the solvent control and were above the laboratory historical control data range. No increases were observed in tester strains TA1535, TA1537, TA98 and WP2uvrA.
To verify the response observed in the first mutation experiment, an additional mutation experiment was performed with tester strain TA100. In the additional direct plate experiment, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Conclusions:

In an Ames test, performed according to OECD 471 guideline and GLP principles, KW-2200 was found not to be mutagenic with or without metabolic activation.

Executive summary:

An Ames test was performed according to OECD 471 guideline and GLP principles.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. KW-2200 did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In tester strain TA100, the test item induced up to 2.3- and 2.2-fold increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively. The increases were more than two-fold the solvent control and were above the laboratory historical control data range. No increases were observed in tester strain WP2uvrA.

In the first mutation experiment, the test item was tested up to concentrations of 5000 μg/plate in the strains TA1535, TA1537 and TA98. KW-2200 did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in all test strains in the absence and presence of S9-mix, except in tester strain TA1535 in the presence of S9-mix. No increases in the number of revertants were observed.

In the second mutation experiment, the test item was tested up to concentrations of 5000 μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction of the bacterial background lawn, was observed in tester strain TA1535, TA1537, TA98 and TA100 in the absence of S9-mix and in tester strain TA98 in the presence of S9-mix. No increases in the number of revertants were observed.

To verify the mutagenic response observed in tester strain TA100 in the absence and presence of S9-mix in the dose-range finding a third experiment was performed. In the third experiment, the test item was tested up to concentrations of 5000 μg/plate in the tester strain TA100. The test item did no precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in the absence of S9-mix. No increases 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.

The test item did induce a significant dose-related increase in the number of revertant (His+) colonies in the tester strain TA100 both in the absence and presence of S9-metabolic activation in the first mutation experiment. However, these results could not be confirmed in the second or in the additional experiment. Therefore these increases are considered to be not biologically relevant.

All other bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.

In conclusion, based on the results of this study it is concluded that KW-2200 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 July 2018 - 25 September 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)

Version / remarks:

29 July 2016

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):

CELLS USED
- Cell cycle length, doubling time or proliferation index: Average Generation Time (AGT): 13.5-13.9 h
- Sex, age and number of blood donors if applicable: Blood was collected from healthy adult, non-smoking volunteers (approximately 18 to 35 years of age).
- Whether whole blood or separated lymphocytes were used if applicable: Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin (Vacuette, Greiner Bio-One, Alphen aan den Rijn, The Netherlands). Immediately after blood collection lymphocyte cultures were started.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Culture medium consisted of RPMI 1640 medium (Life technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life technologies), L-glutamine (2 mM) (Life technologies), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) (Life technologies) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands). All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 61 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.1 - 37.3°C).
- 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.

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:

Dose range finding test:
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 1.0, 2.0, 3.9, 7.8, 15.6 and 31.3 μg/mL
First cytogenetic test:
Without and with S9-mix, 3 h exposure time, 24 h fixation time: 3.9, 7.8 and 15.6 μg/mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 3.9, 7.8 and 15.6 μg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 7.8, 15.6 and 31.3 μg/mL

Vehicle / solvent:

- Vehicle used: culture medium
- Justification for choice of vehicle: A correction factor of 1.13 for the purity/composition of the test item was applied in this study. A solubility test was performed based on visual assessment. The test item formed a pink homogenous suspension in culture medium at concentrations of 50 mg/mL and lower. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. Test item concentrations were used within 2 hours after preparation.

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 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

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 μg/mL medium) (Acros Organics, Geel, Belgium). Thereafter the cell cultures were centrifuged for 5 min at 365 g and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride (Merck) solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol (Merck): acetic acid (Merck) fixative (3:1 v/v).
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 150 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: The mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells (with a maximum deviation of 5%).

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

Evaluation criteria:

A test item is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.

Key result

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
- Precipitation: The test item precipitated at concentrations of 15.6 μg/mL and upwards.
-
RANGE-FINDING/SCREENING STUDIES: No cytotoxicity was observed in the duplicate cultures of the 24 h and 48 h exposure times.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
3 hours exposure time 24 hours exposure time 48 hours exposure time
Gaps included Gaps excluded Gaps included Gaps excluded Gaps included Gaps excluded
+ S9-mix - S9-mix + S9-mix - S9-mix - S9-mix - S9-mix - S9-mix - S9-mix
Mean number of aberrant cells per 100 cells 21.63 24.16 20.78 23.65 26.76 26.09 33.65 32.92
SD 10.45 12.83 10.26 13.04 14.43 14.41 15.28 15.26
n 128 130 128 130 124 124 120 120
Upper control limit
(95% control limits) 39.56 44.96 38.16 44.70 51.64 50.92 63.20 62.08
Lower control limit
(95% control limits) 3.70 3.35 3.39 2.60 1.89 1.25 4.10 3.76
SD = Standard deviation
n = Number of observations
Distribution historical positive control data from experiments performed between September 2015 and September 2018.

- Negative (solvent/vehicle) historical control data:
3 hours exposure time 24 hours exposure time 48 hours exposure time
Gaps included Gaps excluded Gaps included Gaps excluded Gaps included Gaps excluded
+ S9-mix - S9-mix + S9-mix - S9-mix - S9-mix - S9-mix - S9-mix - S9-mix
Mean number of aberrant cells per 100 cells 0.40 0.50 0.31 0.42 0.44 0.37 0.87 0.69
SD 0.53 0.73 0.45 0.68 0.65 0.63 2.44 2.28
n 130 130 130 130 126 126 122 122
Upper control limit
(95% control limits) 1.68 2.32 1.39 1.95 1.92 1.71 3.92 3.13
Lower control limit
(95% control limits) -0.89 -1.32 -0.77 -1.10 -1.03 -0.96 -2.17 -1.74
SD = Standard deviation
n = Number of observations
Distribution historical negative control data from experiments performed between September 2015 and September 2018.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxicity was observed in the duplicate cultures treated with the substance of the 3 h, 24 h and 48 h exposure times.

Conclusions:

A chromosome aberration study with KW-2200 was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that KW-2200 is not clastogenic in human lymphocytes.

Executive summary:

A chromosome aberration study with KW-2200 was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments.

No toxicity was observed at the highest tested precipitating concentration of 31.3 μg/mL. Both in the absence and presence of S9-mix KW-2200 did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

No effects of KW-2200 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 KW-2200 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Based on the results it can be concluded that KW-2200 is not clastogenic in human lymphocytes.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 November 2018 - 18 December 2018

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 gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and 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

For cell lines:
- Absence of Mycoplasma contamination: Yes
- Methods for maintenance in cell culture: stock cultures of the cells were stored in liquid nitrogen (-196°C).
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Basic medium:
RPMI 1640 Hepes buffered medium (Dutch modification) (Life Technologies) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively) (Life Technologies), 1 mM sodium pyruvate (Sigma, Zwijndrecht, The Netherlands) and 2 mM L-glutamin (Life Technologies).
Growth medium:
Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Exposure medium
For 3 hour exposure:
Cells were exposed to the test item in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).
For 24 hour exposure:
Cells were exposed to the test item in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
Selective medium:
Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 µg/mL trifluorothymidine (TFT) (Sigma).
Non-selective medium:
Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium).
Environmental conditions:
All incubations were carried out in a humid atmosphere (80 - 100%, actual range 69 - 99%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.4 - 37.3 °C).

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared 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:

- Dose-range finding test, with S9 (3h) and without S9 (3h and 24h): 0.78, 1.6, 3.1, 6.3 and 12.5 μg/mL
- Experiment 1, with and without S9 (3h): 0.05, 0.10, 0.20, 0.39, 0.78, 1.6, 3.1 and 6.3 μg/mL
- Experiment 2, without S9 (24h): 0.02, 0.05, 0.1, 0.2, 0.39, 0.78, 1.6 and 3.1 μg/mL.

Vehicle / solvent:

- Solvent used: aqueous solvents (water or saline or culture medium)]
- Justification for choice of solvent: A solubility test was performed based on visual assessment. The test item formed a pink/orange homogenous suspension in RPMI 1640 (exposure medium (R5)).
The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. A correction factor of 1.13 for the purity/composition of the test item was applied in this study.
- Justification for percentage of solvent in the final culture medium: The final concentration of the solvent in the exposure medium was 1% (v/v).

Negative solvent / vehicle controls:

yes

Remarks:

RPMI 1640 (exposure medium (R5)

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) :
test concentrations: 1 per test concentration; positive control: 1; solvent control: 2
- Number of independent experiments : two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): below 1 x 10^6 cells/mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
Short term treatment: 3 hours (with and without S9-mix)
Prolonged treatment: 24 hours (without S9-mix)

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days in which at least 4 x 10^6 cells were subcultured every day
- Selection time (if incubation with a selective agent): 11 or 12 days
- Method used: microwell plates
- Selective agent used: 5 μg/mL trifluorothymidine (TFT)
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
For determination of the mutation frequency (MF) a total number of 9.6 x 10^5 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10^5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection).
- Criteria for small (slow growing) and large (fast growing) colonies: The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.

METHODS FOR MEASUREMENT 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).

Statistics:

Not performed.

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: After 3 and 24 hours, the test item precipitated in the exposure medium at concentrations of 6.3 and 3.1 μg/mL and above, respectively.

RANGE-FINDING/SCREENING STUDIES:
- The test item precipitated in the exposure medium at the test item concentration of 3.1 μg/mL and above.
- 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 12.5 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 12.5 μg/mL compared to the solvent control.

STUDY RESULTS
- Concurrent vehicle negative and positive control data :
The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.
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.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: No significant toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Negative (solvent/vehicle) historical control data:
Mutation frequency per 10^6 survivors
- S9-mix + S9-mix
3 hour treatment 24 hour treatment 3 hour treatment
Mean 101 98 100
SD 30 31 30
n 279 262 293
Upper control limit
(95% control limits) 170 162 165
Lower control limit
(95% control limits) 31 34 36
SD = Standard deviation
n = Number of observations
Distribution historical negative control data from experiments performed between September 2015 and September 2018.

- Positive historical control data:
Mutation frequency per 10^6 survivors
- S9-mix + S9-mix
3 hour treatment 24 hour treatment 3 hour treatment
Mean 803 695 1545
SD 253 223 887
n 142 132 151
Upper control limit
(95% control limits) 1533 1270 3954
Lower control limit
(95% control limits) 72 119 864
SD = Standard deviation
n = Number of observations
Distribution historical positive control data from experiments performed between September 2015 and September 2018

Conclusions:

KW-2200 is not mutagenic in the TK mutation test system.

Executive summary:

A gene mutation test in mammalian cells was performed according to OECD guideline 490 and GLP principles, to assess the potential of KW-2200 to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells.

In the first experiment, KW-2200 was tested up to concentrations of 6.3 µg/mL in the absence and presence S9-mix. The incubation time was 3 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. KW-2200 precipitated in the culture medium at this dose level.

In the second experiment, KW-2200 was tested up to concentrations of 3.1 µg/mL in the absence of S9-mix. The incubation time was 24 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. KW-2200 precipitated in the culture medium at this dose level.

No toxicity was observed up to and including the highest dose level, in the absence and in the presence of S9. Reliable negative and positive controls were included. In the absence and presence of S9-mix, KW-2200 did not induce a biologically relevant increase in the mutation frequency. In conclusion, KW-2200 is not mutagenic in the mouse lymphoma L5178Y test system.

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 471 guideline and GLP principles.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. KW-2200 did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In tester strain TA100, the test item induced up to 2.3- and 2.2-fold increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively. The increases were more than two-fold the solvent control and were above the laboratory historical control data range. No increases were observed in tester strain WP2uvrA.

In the first mutation experiment, the test item was tested up to concentrations of 5000 μg/plate in the strains TA1535, TA1537 and TA98. KW-2200 did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in all test strains in the absence and presence of S9-mix, except in tester strain TA1535 in the presence of S9-mix. No increases in the number of revertants were observed.

In the second mutation experiment, the test item was tested up to concentrations of 5000 μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction of the bacterial background lawn, was observed in tester strain TA1535, TA1537, TA98 and TA100 in the absence of S9-mix and in tester strain TA98 in the presence of S9-mix. No increases in the number of revertants were observed.

To verify the mutagenic response observed in tester strain TA100 in the absence and presence of S9-mix in the dose-range finding a third experiment was performed. In the third experiment, the test item was tested up to concentrations of 5000 μg/plate in the tester strain TA100. The test item did no precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in the absence of S9-mix. No increases 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.

The test item did induce a significant dose-related increase in the number of revertant (His+) colonies in the tester strain TA100 both in the absence and presence of S9-metabolic activation in the first mutation experiment. However, these results could not be confirmed in the second or in the additional experiment. Therefore these increases are considered to be not biologically relevant.

All other bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.

In conclusion, based on the results of this study it is concluded that KW-2200 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Chromosome aberration test:

A chromosome aberration study with KW-2200 was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments.

No toxicity was observed at the highest tested precipitating concentration of 31.3 μg/mL. Both in the absence and presence of S9-mix KW-2200 did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

No effects of KW-2200 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 KW-2200 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Based on the results it can be concluded that KW-2200 is not clastogenic in 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 KW-2200 to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells.

In the first experiment, KW-2200 was tested up to concentrations of 6.3 µg/mL in the absence and presence S9-mix. The incubation time was 3 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. KW-2200 precipitated in the culture medium at this dose level.

In the second experiment, KW-2200 was tested up to concentrations of 3.1 µg/mL in the absence of S9-mix. The incubation time was 24 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. KW-2200 precipitated in the culture medium at this dose level.

No toxicity was observed up to and including the highest dose level, in the absence and in the presence of S9. Reliable negative and positive controls were included.In the absence and presence of S9-mix, KW-2200 did not induce a biologically relevant increase in the mutation frequency.In conclusion, KW-2200 is not mutagenic in the mouse lymphoma L5178Y test system.

Justification for classification or non-classification

Based on the results of the Ames test, in vitro chromosome aberration and mouse lymphoma studies, the substance KW-2200 does not have to be classified for genotoxicity in accordance with Regulation (EC) No 1272/2008 and its amendments.