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Toxicological information

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:
19 September 2018 (Study Initiation) to 22 November 2018 (Study Completion)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test materia: Nitika Pharmaceutical Specialities Pvt. Ltd.
- Lot/batch No.of test material: MNST9H122A
- Expiration date of the lot/batch: May 2023
- Purity test (release) date: 12 June 2018 (CoA)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (ambient), kept in original container as supplied by the Sponsor. Container kept tightly closed in a dry, cool and well ventilated place
- Stability under test conditions: Assumed stable for the duration of the study
- Solubility and stability of the test substance in the solvent/vehicle: Formed a suspension in 95% ethanol. Assumed stable for the duration of the study.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Prepared as a suspension in 95% ethanol prior to dosing

FORM AS APPLIED IN THE TEST (if different from that of starting material) Prepared as a suspension in 95% ethanol prior to dosing

OTHER SPECIFICS:
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: pH and osmolality not specified. No precipitate observed at 5000 µg/plate, the limit guideline concentration, in the absence and presence of metabolic activation.

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : The Salmonella typhimurium strains used in this study were mutants derived from Salmonella typhimurium LT2. The strains used were obtained from Molecular Toxicology Inc., 157 Industrial Park Dr. Boone, NC 28607, USA.

- method of preparation of S9 mix

The prepared co-factor mix was dispensed and stored below 0 °C.
S9 mix (10 mL volume)
Constituent Initial Toxicity Mutation Test Confirmatory Mutation Test
5% v/v S9 mix (10 mL) 10% v/v S9 mix (10 mL)
Co-factor mix 9.5 mL 9.0 mL
S9 fraction 0.5 mL 1.0 mL
The S9 mix was prepared fresh by adding the required quantity of S9 fraction to thawed co-factors and maintained in an ice bath. Any remaining mix was discarded.

- concentration or volume of S9 mix and S9 in the final culture medium. A volume of 0.1 mL of S9 mix (5% v/v S9 mix for the initial toxicity-mutation assay and 10% v/v S9 mix for the confirmatory mutation assay) prepared for treatment was added aseptically to 2 mL of top agar, mixed and poured onto MGA plates.

- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)

Sterility Check For Initial Toxicity Mutation Test Confirmatory Mutation Test
Top agar Sterile Sterile
S9 mix Sterile # Sterile ##
Solvent (95% Ethanol) Sterile Sterile
T.I. Stock Solution * Sterile Sterile
MGA Plate (Blank) Sterile Sterile
ONB Solution Sterile Sterile
0.2M Sodium Phosphate Buffer Sterile Sterile

Key: * = Highest Concentration of Test Item Stock Solution, # = 5% v/v S9 mix, ## =10% v/v S9 mix, T.I. = Test Item, DW = Distilled Water,
MGA = Minimal Glucose Agar, ONB = Oxoid Nutrient Broth
Test concentrations with justification for top dose:
In an initial toxicity-mutation test, bacterial cultures were exposed to Fatty acids, C16-18 (even numbered), manganese(II) salts at 8 concentrations from 1.5 to 5000 µg/plate, the limit guideline concentration. Normal growth and no biologically relevant increase in the number of revertant colonies (i.e. no mutagenic effect) was observed in the absence and presence of metabolic activation in the absence and presence of metabolic activation (5% v/v S9 mix) in tester strains TA1537, TA1535, TA98, TA100, and TA102.
Based on the initial toxicity mutation test, the Confirmatory Mutation Test was performed in tester strains TA1537, TA1535, TA98, TA100, and TA102 upto the limit guideline concentration of 5000 µg/plate using a modified dose spacing and an increased concentration of S9 mix. The selected test concentrations were 156.25, 312.5, 625, 1250, 2500 and 5000 µg/plate of Fatty acids, C16-18 (even numbered), manganese(II) salts in the absence and presence of metabolic activation (10% v/v S9 mix).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 95% ethanol

- Justification for choice of solvent/vehicle: The test item was insoluble in distilled water, DMSO and acetone at 50000 µg/mL, however it formed a suspension in 95% ethanol at 50000 µg/mL. 95% ethanol was therefore selected as the vehicle for treatment. A volume of 100 µL (5000 µg/plate) was added to 2 mL of top agar to assess precipitation. No precipitation was observed at 5000 µg/plate. Therefore, 5000 µg/plate was selected as the highest tested concentration for the initial toxicity-mutation test in the absence and presence (5% v/v S9 mix) of metabolic activation.

- Justification for percentage of solvent in the final culture medium: 5% v/v (0.1 mL of ethanol was used as the vehicle into 2 ml top agar)
Controls
Untreated negative controls:
yes
Remarks:
0.2 M phosphate buffer
Negative solvent / vehicle controls:
yes
Remarks:
95% ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-aminoanthracene +S9 (10 ug/plate: TA1537, TA1535 and TA102). -S9 (5 ug/plate TA98 and TA100)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate plates/concentration in the initial toxicity-mutation test
Triplicate plates/concentration in the confirmatory mutation test.

- Number of independent experiments : Two, an initial toxicity-mutation test and an confirmatory mutation test

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Approximately 1 - 2 x 109 bacteria/mL, demonstrating appropriate bacterial numbers were plated.
- Test substance added in medium; in agar (plate incorporation); Treatments were performed using the plate incorporation technique in all tests.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Petri plates were incubated at 37 ± 1 °C for 48 hours and then examined to assess the background lawn inhibition and reduction in number of colonies.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: Six analysable doses were available to evaluate assay data. Cytotoxicity was detectable as decrease in the number of revertant colonies per plate and/or by a thinning of the bacterial background lawn (Teo et al., 2003).

METHODS FOR MEASUREMENTS OF GENOTOXICIY
A result is considered positive if a concentration-related increase over the range tested and/or a reproducible increase at one or more test concentration in the number of revertant colonies per plate in at least one strain with or without metabolic activation.
Rationale for test conditions:
The study was performed in accordence with the specified experimental conditions as per OECD 471 and EU METHOD B.13/14. This guidence is accepted by the OECD and other regulatory authorities
Evaluation criteria:
A result was considered positive if a concentration-related increase a over the range tested and/or a reproducible increase at one or more test concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation.
The biological relevance of any result was considered.

Strains TA1535, 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 negative control value.

Strains TA98, TA100 and TA102: 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 negative control value.

Statistical analysis was used as an aid in the evaluation of any dose response.

A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) was not evaluated as positive.

Negative results obtained in the initial toxicity-mutation test (II) were confirmed by a confirmatory mutation test using the same method as specified above, with an alteration in concentration spacing and concentration of S9 mix.
Statistics:
A simple linear regression analysis was performed for TA1537, TA1535, TA98, TA100 and TA102, separately, to assess the dose dependent nature of any increase in revertant colonies

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: Not specified
- Data on osmolality: Not specified
- Possibility of evaporation from medium: Non-volatile
- Water solubility: Formed a suspension in 95% ethanol at 50 mg/mL (stock solution). Treated a suspension (01 mL/2 mL plate) to acheive the limit guideline concentration of 5000 ug/plate
- Precipitation and time of the determination: No precipitate observed at 5000 µg/plate

STUDY RESULTS
- Concurrent vehicle negative and positive control data : All values for the negative control (95% ethanol) in all strains were comparable with the untreated control of respective strains and also with the laboratory historical data of distilled water and other acceptable solvents, indicating that the solvent does not have any impact on bacterial growth. Positive controls showed a clear increase in the number of revertant colonies demonstrating the efficiency of the test system.

Ames test:
- Signs of toxicity : None
- Individual plate counts : yes
- Mean number of revertant colonies per plate and standard deviation ; yes

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: yes
- Negative (solvent/vehicle) historical control data: no, as an non-standard vehicle (95% ethanol) was used. All values for the negative control (95% ethanol) in all strains were comparable with the untreated control of respective strains and also with the laboratory historical data of distilled water and other acceptable solvents, indicating that the solvent does not have any impact on bacterial growth.

Applicant's summary and conclusion

Conclusions:
It was concluded that Fatty acids, C16-18 (even numbered), manganese(II) salts is non-mutagenic to any of the five strains of Salmonella typhimurium viz., TA1537, TA1535, TA98, TA100 and TA102 when tested under the specified experimental conditions.