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Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
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

Test material

Constituent 1
Reference substance name:
Reaction products of fatty acids, C18-unstaturated, dimers and trimers with 3,3'-[oxybis(ethane-2,1-diyloxy)]dipropan-1-amine
EC Number:
701-270-9
Cas Number:
68911-25-1
Molecular formula:
N.A. UVCB substance
IUPAC Name:
Reaction products of fatty acids, C18-unstaturated, dimers and trimers with 3,3'-[oxybis(ethane-2,1-diyloxy)]dipropan-1-amine
Test material form:
liquid
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.

Method

Target gene:
Histidine and tryptophan operon
Species / strainopen allclose all
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.
Controls
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.

Results and discussion

Test resultsopen allclose all
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.

Applicant's summary and conclusion

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.