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Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genotoxicity/mutagenicity potential of MDP was evaluated in an in vitro assay. The result of the study was:

When tested according to OECD 471: Negative in the presence and absence of metabolic activation.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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:

1997

Deviations:

no

Remarks:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: 3M Company, Lot 668854.
- Purity, including information on contaminants, isomers, etc.: 97.2%, reaction mass as described in the general information section.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 20-25 C, protected from light.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: No data
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: No data
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: No data
- Reactivity of the test material with the incubation material used (e.g. plastic ware): No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): The test article was suspended in DMSO.

FORM AS APPLIED IN THE TEST: Suspended in DMSO.

Target gene:

Tryptophan operon, histidine operon.

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Aroclor-induced rat liver S9 fraction.
- source of S9 : The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL of S9 was added to each plate
- quality controls of S9: Sterility and activity were confirmed. Activity was evaluated with benzo(a)pyrene and 2-aminoanthracene.

Test concentrations with justification for top dose:

In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate. Precipitate was observed beginning at 3333 µg per plate with all conditions. Toxicity was observed beginning at 3333 or at 5000 µg per plate with tester strains TA1537 and WP2 uvrA in the presence of S9 activation. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 µg per plate. In the mutagenicity assay, the dose levels tested were 33.3, 100, 333, 1000, 3333 and 5000 µg per plate. Precipitate was observed at 5000 µg per plate with all conditions. Toxicity was observed beginning at 3333 or at 5000 µg per plate with tester strains TA100, TA1535 and WP2 uvrA in the absence of S9 activation.

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: Test article solubility and assay compatibility.

- Justification for percentage of solvent in the final culture medium: Per OECD 471 guideline.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of greater than or equal to 0.3x109 cells per milliliter.
- Test substance added in medium; in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a shaker/incubator programmed to begin shaking at 125 to 175 rpm and incubating at 37±2°C for approximately 12 hours before the anticipated time of harvest.
- Exposure duration/duration of treatment: 48-72 hours
- Harvest time after the end of treatment (sampling/recovery times): 48-72 hours following the start of exposure.

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 48-72 hours
- Selection time (if incubation with a selective agent): 48-72 hours in typtophan and histidine-minimal agar
- Fixation time (start of exposure up to fixation or harvest of cells): 48-72 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method.: Microcology lawn health
- Any supplementary information relevant to cytotoxicity: A minimum of three non-toxic dose levels is required to evaluate assay data. A dose level is considered toxic if one or both of the following criteria are met: (1) A >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) At least a moderate reduction in the background lawn.

METHODS FOR MEASUREMENTS OF GENOTOXICIY

For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article as specified below:

Strains TA1535 and TA1537
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.

Strains TA98, TA100 and WP2 uvrA
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.

Rationale for test conditions:

Per OECD 471

Evaluation criteria:

See 'Methods for Measurements of Genotoxicity' field.

Statistics:

None, calculations were made to determine if 2-3 fold increases in revertants were observed compared to controls.

Key result

Species / strain:

S. typhimurium TA 98

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

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation was observed at 5000 ug/plate with and without metabolic activation.

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed at 5000 ug/plate without metabolic activation. Precipitation was observed at 5000 ug/plate with and without metabolic activation.

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Cytotoxicity was observed at 5000 ug/plate with metabolic activation. Precipiation was observed at 5000 ug/plate with and without metabolic activation.

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipliation was observed at 5000 ug/plate with and without metabolic activation.

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:
- Data on osmolality:
- Possibility of evaporation from medium:
- Water solubility:
- Precipitation and time of the determination:
- Definition of acceptable cells for analysis:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES (if applicable):

STUDY RESULTS
- Concurrent vehicle negative and positive control data

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value if any
- Any other criteria: e.g. GEF for MLA

Ames test:
- Signs of toxicity
- Individual plate counts
- Mean number of revertant colonies per plate and standard deviation

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For lymphocytres in primary cultures: mitotic index (MI)
o For cell lines: relative population doubling (RPD), relative Increase in cell count (RICC), number of cells treated and cells harvested for each culture, information on cell cycle length, doubling time or proliferation index.
- Genotoxicity results (for both cell lines and lymphocytes)
o Definition for chromosome aberrations, including gaps
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excludling gaps
o Changes in ploidy (polyploidy cells and cells with endoreduplicated chromosomes) if seen

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
o In the case of the cytokinesis-block method: CBPI or RI; distribution of mono-, bi- and multi-nucleated cells
o When cytokinesis block is not used: RICC, RPD or PD, as well as the number of cells treated and of cells harvested for each culture
o Other observations when applicable (complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells)

- 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

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures
o Number of cells plated in selective and non-selective medium
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency
o When using the thymidine kinase gene on L5178Y cells: colony sizing for the negative and positive controls and if the test chemical is positive, and related mutant frequency. For the MLA, the GEF evaluation.

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:
- Negative (solvent/vehicle) historical control data:

Conclusions:

Based on the results of the study, MDP was negative in the bacterial reverse mutation assay (Ames assay) when tested in S. typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli strain WP2 uvrA in the presense and absence of metabolic activation (S9).

Executive summary:

The mutagenic potential of MDP was evaluated in the bacterial reverse mutation assay (Ames assay). The study was conducted according to OECD 471 in compliance with OECD GLP. DMSO was the vehicle used to deliver the test article (MDP) to the test system via the plate incorporation method. In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate.  Precipitate was observed beginning at 3333 µg per plate with all conditions. Toxicity was observed beginning at 3333 or at 5000 µg per plate with tester strains TA1537 and WP2 uvrA in the presence of S9 activation. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 µg per plate. In the mutagenicity assay, the dose levels tested were 33.3, 100, 333, 1000, 3333 and 5000 µg per plate. Precipitate was observed at 5000 µg per plate with all conditions. Toxicity was observed beginning at 3333 or at 5000 µg per plate with tester strains TA100, TA1535 and WP2 uvrA in the absence of S9 activation. All validation criteria was met for the study and vehicle and positive controls performed as expected. In the plates treated with MDP, there were no increases in revertant colonies in any tester strains, at any concentration with or without metabolic activation. Based on the results of the study, MDP was negative in the bacterial reverse mutation assay (Ames assay) when tested in S. typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli strain WP2 uvrA in the presence and absence of metabolic activation (S9).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the results of the study, MDP does not meet the GHS Criteria for classification for genotoxicity/mutagenicity.