Reaction product of Fatty acids, C18-unstaturated, dimer with 4,7,10-trioxa-1,13-tridecane-diamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro studies were conducted on MTDID 18990. The results of the studies were:

When tested according to OECD 471: Negative with and without metabolic activation.

When tested according to OECD 487: Negative with and without metabolic activation.

When tested according to OECD 490: Negative with and without metabolic activation.

The objective of this study was to determine the potential of MTDID 18990 and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9). The study was conducted according to OECD 471 in compliance with OECD GLP. The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay. MTDID 18990 was a clear to cloudy dark brown-green liquid. The vehicle of the test item was dimethyl sulfoxide. 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. MTDID 18990 precipitated on the plates at the top dose level of 5000μg/plate. Since the test item showed moderate precipitate on the plates at the test item concentration of 5000 μg/plate, thebacterial background of this dose level could not be determined. Cytotoxicity, as evidenced by a decrease in the number of revertants and a reduction of the bacterial background lawn, was observed in tester strain TA100 strain in the absence and presence of S9-mix and by a decrease in the number of revertants in testers train WP2uvrAin the presence of S9-mix. Results of this dose-range finding test were reported as part of the first mutation assay. In the first mutation experiment, the test item was tested up to concentrations of 5000 μg/plate in the strains TA1535, TA1537 and TA98. MTDID 18990 precipitated on the plates at the top dose level of 5000 μg/plate.Since the test item showed moderate precipitate on the plates at the test item concentration of 5000 ug/plate, thebacterial background of this dose level could not be determined for tester strains TA1537 and TA98. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix. 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. MTDID 18990 precipitated on the plates at the top dose level of 5000 μg/plateexcept in tester strain TA100 in the presence of S9-mix where precipitate was observed at dose levels of 1600 μg/plate and upwards. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix. 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 not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. Based on the results of this study, MTDID 18990 is not mutagenic in the Ames assay in the presence or absence of metabolic activation.

The ability for MTDID 18990 to induce micronuclei in culture human lymphocytes was evaluated in the presence and absence of metabolic activation (S9 -mix). The study was conducted according to OECD 487 in compliance with OECD GLP. The test article was suspended in DMSO for dosing. Lymphocytes (0.4 mL blood of a healthy donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 46 ± 2 hours and thereafter exposed to selected doses of MTDID 18990 for 3 hours and 24 hours in the absence of S9-mix or for 3 hours in the presence of S9-mix. Cytochalasine B (Sigma) was added to the cells simultaneously with the test item at the 24 hours exposure time. A vehicle control was included at each exposure time. The highest tested concentration was determined by the solubility of MTDID 18990 in the culture medium. The test item precipitated at concentrations of 39 μg/mL and upwards. The lymphocytes were cultured in duplicate at the 3 h exposure time and appropriate positive controls were included. After 3 hours exposure to MTDID 18990 in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium with Cytochalasine B (5 μg/mL) and incubated for another 24 hours (1.5 times normal cell cycle). The cells that were exposed for 24 hours in the absence of S9-mix were not rinsed after exposure but were fixed immediately. Cytotoxicity of MTDID 18990 in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index). No cytotoxicity was observed in the duplicate cultures of the 3 h exposure time and the slides were scored for micronuclei. The pilot study (short term exposure period) was used as the first cytogenetic assay. Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the second cytogenetic assay considering the highest dose level was determined by the solubility. To confirm the results of the first cytogenetic assay a second cytogenetic assay was performed with an extended exposure time of the cells in the absence of S9-mix. Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of MTDID 18990 with cytochalasin B (5 μg/mL) for 24 hours in the absence of S9-mix. Appropriate vehicle and positive controls were included in the second cytogenetic assay. To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were re-suspended in 1% Pluronic F68 (Applichem, Darmstadt, Germany). After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride (Merck) solution. Immediately after, ethanol (Merck): acetic acid (Merck) fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid 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 6.7% (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). A minimum of 500 cells (with a maximum deviation of 5%) per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells). The cytostasis / cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI). Three analyzable concentrations were scored for micronuclei. The number of micronuclei per cell was not recorded. MTDID 18990 was not cytotoxic and/or difficult to dissolve in aqueous solutions, the highest concentration analyzed was determined by the solubility in the culture medium. At least 1000 (with a maximum deviation of 5%) binucleated cells per culture were  examined by light microscopy for micronuclei. In addition, at least 1000 (with a maximum deviation of 5%) mononucleated cells per culture were scored for micronuclei separately.  MTDID 18990 did not induce a statistically significantly different CBPI at any concentration compared to the vehicle control.  Precipitation was observed at MTDID 18990 concentrations of 39 ug/mL and above.  The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control  chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition colchicine also showed a statistically significant increase in the number of binucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei 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. MTDID 18990 did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

The objective of this study was to evaluate the mutagenic potential of MTDID 18990 by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions. The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.  The study was conducted according to OECD 490 in compliance with OECD GLP.  MTDID 18990 was suspended in DMSO for dosing.   Per culture 8 x 10^6 cells (106 cells/mL for 3 hour treatment) or 6 x 10^6  cells (1.25 x 10^5 cells/mL for 24 hour treatment) were used. The cell cultures for the 3 hour treatment were placed in sterile 30 mL centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 rpm. The cell cultures for the 24 hour treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. Solvent and positive controls were included and the solvent control was tested in duplicate. In the first experiment, cell cultures were exposed for 3 hours to MTDID 18990 in exposure medium in the absence and presence of S9-mix. In the second experiment, cell cultures were exposed to MTDID 18990 in exposure medium for 24 hours in the absence of S9-mix. For the 3 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 50 mL growth medium (R10-medium). For the 24 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 20 mL growth medium (R10-medium). The cells in the final suspension were counted with the coulter particle counter.  For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 10^6 cells (where possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test item the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF). 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). The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 1.5-2.0 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope. The colonies were divided into small and large colonies. Mutant cells that have suffered extensive genetic damage have prolonged doubling times and thus form small colonies. Less severely affected mutant cells grow at rates similar to the parental cells and form large colonies. The small colonies can be associated with the induction of chromosomal mutations. The large colonies appear to result from mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene. 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. In the first experiment, MTDID 18990 was tested up to concentrations of 50 μg/mL in the absence and presence S9-mix. The incubation time was 3 hours. The Relative total growth (RTG) was reduced to 43% in the absence of S9-mix. No toxicity was observed at this dose level in the presence of S9-mix. MTDID 18990 precipitated in the culture medium at this dose level. In the second experiment, MTDID 18990 was tested up to concentrations of 30 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 9%. 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. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. In the absence of S9-mix, MTDID 18990 did not induce a biologically relevant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, MTDID 18990 did not induce a biologically relevant increase in the mutation frequency. In conclusion, MTDID 18990 is not mutagenic in the mouse lymphoma L5178Y test system in the presence or 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:

2019

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:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 3M Company, Batch 4293484
- Expiration date of the lot/batch: 31 January, 2021
- Purity test date: 19 December, 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light.
- Stability under storage conditions: No data
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: The test item formed a (yellow) suspension in DMSO. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension.
Test item concentrations were used within 3 hours after preparation.
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable): No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item formed a (yellow) suspension in DMSO. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension.

FORM AS APPLIED IN THE TEST: The test article was suspended in DMSO.

Target gene:

Histidine and tryptophan operon

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:

Type and composition of metabolic activation system:
- source of S9 : Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL Milli-Q water (Millipore Corp., Bedford, MA., USA); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution (Merck); 1 mL 0.33 M KCl solution (Merck). The above solution was filter (0.22 μm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added
(5% (v/v) S9-fraction) to complete the S9-mix.
- concentration or volume of S9 mix and S9 in the final culture medium : 0.5 mL of S9-mix was utilized in the activation assays.
- quality controls of S9: Each S9 batch was characterized with the mutagens benzo-(a)-pyrene (Sigma) and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.

Test concentrations with justification for top dose:

Direct Plate Assay: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate

Pre-incubation Assay: MTDID 18990 was tested up to the dose level of 5000 μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Since severe toxicity was observed in all Salmonella typhimurium strains an additional experiment was performed to complete the data. The following dose range was selected for the additional mutation assay: 0.1, 0.5, 1, 5, 10 and 20 μg/plate in the absence of S9-mix and 5, 10 and 20 μg/plate in the presence of S9-mix in tester strain TA98, TA1535, TA1537 and TA100.

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: The solubility of the test item was evaluated in Study Facility No. 20183638. The test item formed a (yellow) suspension in DMSO.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191, 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments : 2 (the Pre-incubation assay was also run twice)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 10^9 cells/mL
- Test substance exposure method: A direct plate assay and a pre-incubation assay were conducted.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Direct Plate Assay: None, Pre-incubation assay: 30 minutes
- Exposure duration/duration of treatment: Direct Plate assay: 48 hours, Pre-incubation assay: 48 hours
- Harvest time after the end of treatment (sampling/recovery times): Direct Plate assay: 48 hours, Pre-incubation assay: 48.5 hours

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and counting): Direct Plate assay: 48 hours, Pre-incubation assay: 48.5 hours
- Fixation time (start of exposure up to fixation or harvest of cells): Direct Plate assay: 48 hours, Pre-incubation assay: 48.5 hours
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 10^9 cells/mL (0.1 mL per plate). The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Bacterial background lawn

METHODS FOR MEASUREMENTS OF GENOTOXICIY : 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.

Rationale for test conditions:

Per OECD 471.

Evaluation criteria:

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

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:

No formal hypothesis testing was done.
In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

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.

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

Direct Plate Assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Moderate precipitation was observed at 5000 ug/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

Direct Plate Assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Moderate cytotoxicity and precipitation was observed at 5000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Direct Plate Assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slight cytotoxicity was observed starting at 512 ug/plate without S9 and at 1600 ug/plate with S9. Moderate precipitation was observed at 5000 ug/plate with and without S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Direct Plate Assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Moderate precipitation was observed at 5000 ug/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

Direct Plate Assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Moderate precipitation was observed at 5000 ug/plate with and without S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

Pre-incubation Assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Extreme cytotoxicity was observed at 17 ug/plate and above with slight cytotoxicity at 10 ug/plate without S9. With S9, extreme cytotoxicity was observed at 1600 and 5000 ug/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

Pre-incubation assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Extreme cytotoxicity was observed at 164 ug/plate and above with moderate cytotoxicity at 52 ug/plate without S9. With S9, extreme cytotoxicity was observed at 1600 and 5000 ug/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Pre-incubation assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Extreme cytotoxicity was observed at 52 ug/plate and above and moderate cytotoxicity at 20 ug/plate without S9 mix. With S9, slight cytotoxicity was noted at 512 ug/plate, moderate at 1600 and extreme at 5000 ug/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Pre-incubation assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without S9, moderate cytotoxcity was observed at 20 ug/plate, slight cytotoxicity at 164 ug/plate, moderate at 512 and extreme at 1600 and 5000 ug/plate. With S9, extreme cytotoxicity was observed at 1600 and 5000 ug/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

Pre-incubation assay.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without and without S9, slight cytotoxicity was observed at 1600 ug/plate and extreme cytotoxicity was observed at 5000 ug/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No data
- Data on osmolality: No data
- Possibility of evaporation from medium: Not expected based on phys-chem properties.
- Water solubility: No data, test article was suspended in DMSO.

RANGE-FINDING/SCREENING STUDIES (if applicable): A range-finding study was conducted to determine the dosing levels for the Direct Plate assay.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Vehicle control and positive control data both performed as expected and were within laboratory historical results indicating that the test system was valid.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : No increase in revertant colonies compared to the vehicle controls was observed.
- Statistical analysis: See 'Statistics' section.

Ames test:
- Signs of toxicity : Bacterial lawn growth was evaluated to determine cytotoxicity. See "Test Results" section for more details.
- Mean number of revertant colonies per plate and standard deviation : The mean number of revertant colonies for each dose group for each strain was comparable or lower than the vehicle control in all assays. No increase in revertants was observed.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Positive controls performed as expected compared to the lab's historical values.
- Negative (solvent/vehicle) historical control data :Vehicle controls performed as expected compared to the lab's historical values.

Conclusions:

Based on the results of this study, MTDID 18990 is not mutagenic in the Ames assay in the presence or absence of metabolic activation.

Executive summary:

The objective of this study was to determine the potential of MTDID 18990 and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9). The study was conducted according to OECD 471 in compliance with OECD GLP. The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay. MTDID 18990 was a clear to cloudy dark brown-green liquid. The vehicle of the test item was dimethyl sulfoxide. 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. MTDID 18990 precipitated on the plates at the top dose level of 5000μg/plate. Since the test item showed moderate precipitate on the plates at the test item concentration of 5000 μg/plate, thebacterial background of this dose level could not be determined. Cytotoxicity, as evidenced by a decrease in the number of revertants and a reduction of the bacterial background lawn, was observed in tester strain TA100 strain in the absence and presence of S9-mix and by a decrease in the number of revertants in testers train WP2uvrAin the presence of S9-mix. Results of this dose-range finding test were reported as part of the first mutation assay. In the first mutation experiment, the test item was tested up to concentrations of 5000 μg/plate in the strains TA1535, TA1537 and TA98. MTDID 18990 precipitated on the plates at the top dose level of 5000 μg/plate.Since the test item showed moderate precipitate on the plates at the test item concentration of 5000 ug/plate, thebacterial background of this dose level could not be determined for tester strains TA1537 and TA98. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix. 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. MTDID 18990 precipitated on the plates at the top dose level of 5000 μg/plateexcept in tester strain TA100 in the presence of S9-mix where precipitate was observed at dose levels of 1600 μg/plate and upwards. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix. 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 not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. Based on the results of this study, MTDID 18990 is not mutagenic in the Ames assay in the presence or absence of metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

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 number of test material: 3M Company, Lot 4293484
- Expiration date of the lot/batch: 31 January 2021
- Purity test date: 19 December, 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
- Stability under storage conditions: No data
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: A solubility test was performed based on visual assessment. The test item formed a clear yellow solution in dimethyl sulfoxide (DMSO, SeccoSolv, Merck, Darmstadt, Germany). Test item concentrations were used within 3 hours after preparation.
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable): No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test article was suspended in DMSO.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Suspended in DMSO

Species / strain / cell type:

lymphocytes: See Remarks

Remarks:

Cultured peripheral human lymphocytes were utilized as recommended in the international OECD 487 guideline.

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Cultured peripheral human lymphocytes from healthy adult, non-smoking volunteers (18-35).
- Suitability of cells: Recommended per OECD 478.
- Normal cell cycle time (negative control): The average generation time (AGT) of the cells for each assay:
Dose-range finding study: age 25, AGT = 12.8 h
First cytogenetic assay: age 25, AGT = 12.8 h
Second cytogenetic assay: age 27, AGT = 12.9 h

For lymphocytes:
- Sex, age and number of blood donors:
Dose-range finding study: age 25
First cytogenetic assay: age 25
Second cytogenetic assay: age 27
- Whether whole blood or separated lymphocytes were used: Whole blood
- Whether blood from different donors were pooled or not: Not pooled
- Mitogen used for lymphocytes: Phytohaemagglutinin.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, 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). Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin (Remel Europe Ltd., Dartford, United Kingdom) was added. All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 55 - 97%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.7 - 36.7°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Cytokinesis block (if used):

A minimum of 500 cells (with a maximum deviation of 5%) per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells). The cytostasis / cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is 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).
- source of S9 : Trinova Biochem
- method of preparation of S9 mix : S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 μmol HEPES (Life Technologies). The above solution was filter (0.22 m)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix. Metabolic activation was achieved by adding 0.2 mL S9-mix to 5.3 mL of a lymphocyte
culture (containing 4.8 mL culture medium, 0.4 mL blood and 0.1 mL (9 mg/mL) phytohaemagglutinin).
- concentration or volume of S9 mix and S9 in the final culture medium : The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).

Test concentrations with justification for top dose:

Without S9, 24 hour exposure time, 24 hour harvest time: 2.4, 4.9, 9.8, 20, 39, 78 ug/mL.
Without S9, 3 hour exposure time, 27 hour harvest time: 9.8, 20, 39 ug/mL.
With S9, 3 hour exposure time, 27 hour harvest time: 9.8, 20, 39 ug/mL.

Dose levels were based on a range-finding study that indicated that the test article precipitated in media starting at 39 ug/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test article formed a homogenous suspension in DMSO.

- Justification for percentage of solvent in the final culture medium: No data

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments : Two

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

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 46 hours
- Exposure duration/duration of treatment: 3 or 24 hours.
- Harvest time after the end of treatment (sampling/recovery times): Without S9, 24 hour exposure time, 24 hour harvest time: 2.4, 4.9, 9.8, 20, 39, 78 ug/mL.
Without S9, 3 hour exposure time, 27 hour harvest time: 9.8, 20, 39 ug/mL.
With S9, 3 hour exposure time, 27 hour harvest time: 9.8, 20, 39 ug/mL.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays):Cytochalasin B, 5 ug/mL were administered at the start of exposure to the test article.
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure.: :Cytochalasin B, 5 ug/mL were administered at the start of exposure to the test article.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were re-suspended in 1% Pluronic F68 (Applichem, Darmstadt, Germany). After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride (Merck) solution. Immediately after, ethanol (Merck): acetic acid (Merck) fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid 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 6.7% (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 cells spread and analysed per concentration: a minimum of 500 cells per culture.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): To prevent bias, all slides were randomly coded before examination of micronuclei and scored. An adhesive label with Charles River Den Bosch study identification number and code was stuck over the marked slide. At least 1000 (with a maximum deviation of 5%) binucleated cells per culture were examined by light microscopy for micronuclei. In addition, at least 1000 (with a maximum deviation of 5%) mononucleated cells per culture were scored for micronuclei separately. Since the lowest concentration of CP resulted in a positive response the highest concentration was not examined for the presence of micronuclei. Due to cytotoxicity the number of examined bi- or mononucleated cells in the positive control groups might be <1000. However, when an expected statistical significant increase was observed,
this has no effect on the study integrity.
The following criteria for scoring of binucleated cells were used (1 - 2, 6):
- Main nuclei that were separate and of approximately equal size.
- Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
- Main nuclei that were linked by nucleoplasmic bridges.
The following cells were not scored:
- Trinucleated, quadranucleated, or multinucleated cells.
- Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).
The following criteria for scoring micronuclei:
- The diameter of micronuclei should be less than one-third of the main nucleus.
- Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
- Micronuclei should have similar staining as the main nucleus.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Cytokinesis-block proliferation index

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.
A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage 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 or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

- OTHER:

Rationale for test conditions:

Per OECD 487

Evaluation criteria:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.
A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage 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 or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage 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.
A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage 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 or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Key result

Species / strain:

lymphocytes: See Remarks

Remarks:

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

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No data
- Data on osmolality: No data
- Possibility of evaporation from medium: Not expected based on the phys-chem properties of the substance.
- Water solubility: No data
- Precipitation and time of the determination: Precipitation was observed at 39 ug/mL and greater concentrations during the 3 and 24 hour exposures.
- Definition of acceptable cells for analysis:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES (if applicable):

STUDY RESULTS
- Concurrent vehicle negative and positive control data
Positive historical control data:
The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition colchicine also showed a statistically significant increase in the number of binucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei 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.
- Negative (solvent/vehicle) historical control data: The number of mono- and binucleated cells with micronuclei found in the solvent control was within the 95% control limits of the distribution of the historical negative control database.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value if any : See 'Statistics' section.

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements: The test article did not induce a statistically significantly different CBPI at any concentration compared to the vehicle control.
o In the case of the cytokinesis-block method: CBPI was utilized. The distribution of mono, bi and tri-nucleiated cells was not statistically significantly different at any concentration compared to the vehicle control.

- Genotoxicity results
o Number of cells with micronuclei separately for each treated and control culture and defining whether from binucleated or mononucleated cells, where appropriate : MTDID 18990 did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data) - Positive historical control data: The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition colchicine also showed a statistically significant increase in the number of binucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei 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.
- Negative (solvent/vehicle) historical control data: The number of mono- and binucleated cells with micronuclei found in the solvent control was within the 95% control limits of the distribution of the historical negative control database.

Conclusions:

MTDID 18990 did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

Executive summary:

The ability for MTDID 18990 to induce micronuclei in culture human lymphocytes was evaluated in the presence and absence of metabolic activation (S9 -mix). The study was conducted according to OECD 487 in compliance with OECD GLP. The test article was suspended in DMSO for dosing. Lymphocytes (0.4 mL blood of a healthy donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 46 ± 2 hours and thereafter exposed to selected doses of MTDID 18990 for 3 hours and 24 hours in the absence of S9-mix or for 3 hours in the presence of S9-mix. Cytochalasine B (Sigma) was added to the cells simultaneously with the test item at the 24 hours exposure time. A vehicle control was included at each exposure time. The highest tested concentration was determined by the solubility of MTDID 18990 in the culture medium. The test item precipitated at concentrations of 39 μg/mL and upwards. The lymphocytes were cultured in duplicate at the 3 h exposure time and appropriate positive controls were included. After 3 hours exposure to MTDID 18990 in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium with Cytochalasine B (5 μg/mL) and incubated for another 24 hours (1.5 times normal cell cycle). The cells that were exposed for 24 hours in the absence of S9-mix were not rinsed after exposure but were fixed immediately. Cytotoxicity of MTDID 18990 in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index). No cytotoxicity was observed in the duplicate cultures of the 3 h exposure time and the slides were scored for micronuclei. The pilot study (short term exposure period) was used as the first cytogenetic assay. Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the second cytogenetic assay considering the highest dose level was determined by the solubility. To confirm the results of the first cytogenetic assay a second cytogenetic assay was performed with an extended exposure time of the cells in the absence of S9-mix. Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of MTDID 18990 with cytochalasin B (5 μg/mL) for 24 hours in the absence of S9-mix. Appropriate vehicle and positive controls were included in the second cytogenetic assay. To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were re-suspended in 1% Pluronic F68 (Applichem, Darmstadt, Germany). After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride (Merck) solution. Immediately after, ethanol (Merck): acetic acid (Merck) fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid 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 6.7% (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). A minimum of 500 cells (with a maximum deviation of 5%) per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells). The cytostasis / cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI). Three analyzable concentrations were scored for micronuclei. The number of micronuclei per cell was not recorded. MTDID 18990 was not cytotoxic and/or difficult to dissolve in aqueous solutions, the highest concentration analyzed was determined by the solubility in the culture medium. At least 1000 (with a maximum deviation of 5%) binucleated cells per culture were  examined by light microscopy for micronuclei. In addition, at least 1000 (with a maximum deviation of 5%) mononucleated cells per culture were scored for micronuclei separately.  MTDID 18990 did not induce a statistically significantly different CBPI at any concentration compared to the vehicle control.  Precipitation was observed at MTDID 18990 concentrations of 39 ug/mL and above.  The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control  chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition colchicine also showed a statistically significant increase in the number of binucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei 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. MTDID 18990 did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

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:

2016

Deviations:

no

Remarks:

No deviations ocurred that impacted the integrity of the study results.

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material:
3M Company, Lot 4293484
- Expiration date of the lot/batch:
31 January 2021
- Purity test date:
19 December, 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
At room temperature protected from light
- Stability under storage conditions:
No data
- Stability under test conditions:
No data
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium:
A solubility test was performed based on visual assessment. The test item formed a clear yellow solution in dimethyl sulfoxide (DMSO, SeccoSolv, Merck, Darmstadt, Germany). Test item concentrations were used within 3 hours after preparation.
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable):
No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The test article was suspended in DMSO.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
: Suspended in DMSO

Target gene:

Thymidine kinase

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 the American Type Culture Collection, (ATCC, Manassas, USA)
(2001).
- Suitability of cells: Recommended per OECD 490.

For cell lines:
- Absence of Mycoplasma contamination: Stock cultures of the cells were stored in the freezer (-150°C). The cultures were checked for mycoplasma contamination. Cell density was kept below 1 x 106 cells/mL.
- Periodically ‘cleansed’ of spontaneous mutants: Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown
for 1 day in R10-medium containing 10-4 M hypoxanthine (Sigma), 2 x 10-7 M aminopterine (Fluka Chemie AG, Buchs, Switzerland) and 1.6 x 10-5 M thymidine (Sigma) (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10-medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment.


MEDIA USED
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 27 - 102%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 32.8 - 47.0 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of 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).
- method of preparation of S9 mix : S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 μmol HEPES (Life technologies). The above solution was filter (0.22 μm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
- concentration or volume of S9 mix and S9 in the final culture medium : The concentration of the S9-fraction in the exposure medium was 4% (v/v).

Test concentrations with justification for top dose:

The test article concentrations were chosen based on a range-finding assay to deterine cytotoxicity.

First Mutagenicity Test:
Without S9-mix: 0.4, 0.8, 1.6, 3.1, 6.3, 12.5, 25, 50, 65 and 75 μg/mL exposure medium.
With S9-mix: 0.8, 1.6, 3.1, 6.3, 12.5, 25, 50 and 75 μg/mL exposure medium.

Second Mutagenicity Test:
Without S9: 0.63, 1.3, 2.5, 5, 10, 15, 20, 30 and 40 μg/mL exposure medium.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:DMSO
- Justification for choice of solvent/vehicle: A solubility test was performed based on visual assessment. The test item formed slightly yellow homogenous suspension in dimethyl sulfoxide.
The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension.
Test item concentrations were used within 1.5 hours after preparation.

- Justification for percentage of solvent in the final culture medium: The final concentration of the solvent in the exposure medium was 1.0% (v/v) which is acceptable per OECD 490.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Single
- Number of independent experiments : Two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Per culture 8 x 10^6 cells (10^6 cells/mL for 3 hour treatment) or 6 x 10^6 cells (1.25 x 10^5 cells/mL for 24 hour treatment) were used.
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: None
- Exposure duration/duration of treatment: 3 or 24 hours
- Harvest time after the end of treatment (sampling/recovery times): Following the exposure periods, for expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 10^6 cells (where possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test item the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF).

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days
- Selection time (if incubation with a selective agent): 11-12 days
- Fixation time (start of exposure up to fixation or harvest of cells): Approximately 13-15 days
- Method used: Microwell plates.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure: 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). The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 1.5-2.0 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: See above
- Criteria for small (slow growing) and large (fast growing) colonies: The colonies were divided into small and large colonies. Mutant cells that have suffered extensive genetic damage have prolonged doubling times and thus form small colonies. Less severely affected mutant cells grow at rates similar to the parental cells and form large colonies. The small colonies can be associated with the induction of chromosomal mutations. The large colonies appear to result from mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene. 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 suspension growth

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The mutation frequency was expressed as the number of mutants per 10^6 viable cells. The plating efficiencies of both mutant and viable cells (CE day2) in the same culture were
determined and the mutation frequency (MF) was calculated as follows:
MF = {-ln P(0)/number of cells plated per well}/ CE day2 x 10^6
Small and large colony mutation frequencies were calculated in an identical manner.

A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(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 MF(controls) + 126.

Rationale for test conditions:

Per OECD 490

Evaluation criteria:

A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) 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 (SG) 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).

A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(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 MF(controls) + 126.

Statistics:

Increased mutation frequency (+126 MF over controls indicating a positive), dose-response and biological relevance were used to evaluate results rather than statistical significance.

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
- Data on pH: No data
- Data on osmolality: No data
- Possibility of evaporation from medium: Not expected based on the phys-chem properties of the substance.
- Water solubility: No data
- Precipitation and time of the determination: The test article precipitated in the exposure medium at concentrations of 75 ug/mL and above in all tests.
- Definition of acceptable cells for analysis: See 'Evaluation Criteria' section.

RANGE-FINDING/SCREENING STUDIES (if applicable): In the dose-range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 9.4 to 150 μg/mL in the absence and presence of S9-mix with a 3 hour treatment periods and with a test item concentration range of 6.3 to 75 μg/mL in the absence of S9-mix with a 24 hour treatment period.
In the absence of S9-mix, the relative suspension growth was 29% at the test item concentration of 37.5 μg/mL compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at test item concentrations of 75 μg/mL and above.
In the presence of S9-mix, the relative suspension growth was 44% at the highest test item concentration of 150 μg/mL compared to the relative suspension growth of the solvent control.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Concurrent vehicle and positive controls performed as expected and were within the historical ranges for the laboratory indicating that the test system was valid and that the metabolic activation system was functioning appropriately.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : No increase in mutant frequency was observed at any exposure concentration.
- Statistical analysis; p-value if any : Increased mutation frequency (+126 MF over controls indicating a positive), dose-response and biological relevance were used to evaluate results rather than statistical significance.
- Any other criteria:GEF = 126

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: In the first test, the test article did not induce severe cytotoxicity but was tested up to precipitating concentrations. In the absence of S9-mix, the relative total growth of the highest precipitating test item concentration was 43% compared to the total growth of the solvent controls. In the presence of S9-mix, no toxicity was observed up to and including the highest (precipitating) tested dose level. Cloning efficiency was similar to vehicle controls for all dose groups.

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures : See 'Details on Test System' section.
o Number of cells plated in selective and non-selective medium : See 'Details on Test System' section.
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency : See 'Details on Test System' for experimental test system details. No increase in mutantion frequency (MF) was overved in any exposure group with or without metabolic activation.
o When using the thymidine kinase gene on L5178Y cells: No biologically relevant increase in the mutation frequency at the TK locus was observed after treatment with the test item. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

HISTORICAL CONTROL DATA
- Positive historical control data: 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.
- Negative (solvent/vehicle) historical 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.

Conclusions:

No biologically relevant increase in the mutation frequency at the TK locus was observed after treatment with MTDID 18990 either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the MTDID 18990 treated cultures were comparable to the numbers of small and large colonies of the solvent controls. In conclusion, MTDID 18990 is not mutagenic in the mouse lymphoma L5178Y test system in the presence or absence of metabolic activation.

Executive summary:

The objective of this study was to evaluate the mutagenic potential of MTDID 18990 by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions. The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.  The study was conducted according to OECD 490 in compliance with OECD GLP.  MTDID 18990 was suspended in DMSO for dosing.   Per culture 8 x 10^6 cells (106 cells/mL for 3 hour treatment) or 6 x 10^6  cells (1.25 x 10^5 cells/mL for 24 hour treatment) were used. The cell cultures for the 3 hour treatment were placed in sterile 30 mL centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 rpm. The cell cultures for the 24 hour treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. Solvent and positive controls were included and the solvent control was tested in duplicate. In the first experiment, cell cultures were exposed for 3 hours to MTDID 18990 in exposure medium in the absence and presence of S9-mix. In the second experiment, cell cultures were exposed to MTDID 18990 in exposure medium for 24 hours in the absence of S9-mix. For the 3 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 50 mL growth medium (R10-medium). For the 24 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 20 mL growth medium (R10-medium). The cells in the final suspension were counted with the coulter particle counter.  For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 10^6 cells (where possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test item the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF). 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). The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 1.5-2.0 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope. The colonies were divided into small and large colonies. Mutant cells that have suffered extensive genetic damage have prolonged doubling times and thus form small colonies. Less severely affected mutant cells grow at rates similar to the parental cells and form large colonies. The small colonies can be associated with the induction of chromosomal mutations. The large colonies appear to result from mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene. 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. In the first experiment, MTDID 18990 was tested up to concentrations of 50 μg/mL in the absence and presence S9-mix. The incubation time was 3 hours. The Relative total growth (RTG) was reduced to 43% in the absence of S9-mix. No toxicity was observed at this dose level in the presence of S9-mix. MTDID 18990 precipitated in the culture medium at this dose level. In the second experiment, MTDID 18990 was tested up to concentrations of 30 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 9%. 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. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. In the absence of S9-mix, MTDID 18990 did not induce a biologically relevant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, MTDID 18990 did not induce a biologically relevant increase in the mutation frequency. In conclusion, MTDID 18990 is not mutagenic in the mouse lymphoma L5178Y test system in the presence or absence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the results of the OECD 471, 487 and 490, MTDID 18990 is not classified as mutagenic.