Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report Date:
2020

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Remarks:
GLP compliance statement is included in attached full study report
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Batch #: 191016
Expiration Date: October 15, 2020
0.9% (9306 mg/L) mineral complexed organic matter (based on total organic carbon content of 0.51%)
1124 mg/L Fe
3333 mg/L Na
2187 mg/L total inorganic carbon
pH=7.28
Density: 1.011 g/mL at 25 degrees C

Method

Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Metabolic Activation System (59 Mix) and Substitution Buffer
S9 mix (cofactor supplemented post-mitochondi-ial fraction) was included in the Ames test to simulate mammalian metabolism since some test substances only become mutagenic following metabolic activatioTi. S9 liver fraction was purchased from Molecular Toxicology. Inc., and sourced from male Sprague-Dawle. rats induced with phenobarbital and 5,6-benzoflavone. The S9 mix, freshly prepared on the day of use, was maintained on ice prior to and during use arid contained 5% v/v 59 fraction. The prepared 59 mix contained the following sterile cofactors (Maron & Ames, 1983): 8 mM MgCI2, 33 mM KCI, 100 mM sodium phosphate buffer pH 7.4, 5 mM glucose-6-phosphate and 4 mM NADP.
Sodium phosphate buffer was used as the substitution buffer for plates treated in the absence of SQ.
Test concentrations with justification for top dose:
No correction for purity was used in preparation of formulations, and all dose levels are expressed in terms of material as supplied.

During initial sterility testing, the test substance was found to have contamination. Therefore, the test substance was sent to VPT Rad Radiation Lab and Test Services, Chelmsford, MA 01824 for sterilization by gamma irradiation at dose 5OkGy prior to testing. The sterilization report is in Appendix B. No contamination was noted after sterilization.

The test substance was formulated as a solution in sterile water (0.0158, 0.05, 0.158. 0.5, 1.58, 5, 15.8 and 50 mg/mL) to provide corresponding dose levels of up to 5000 ig/plate. The solutions were vortexed prior to use.

The Ames test was conducted with FBS Transit at levels of 1.58, 5.0, 15.8, 50, 158, 500, 1580, and 5000 1g/plate, with the high level being the standard limit for this test. The main test was conducted using the plate incorporation method in both the absence and presence of metabolic activation (chemically-induced rat liver S9 mix). The results of the test were confirmed using a similar study design but employing the pre-incubation modification of the Ames test.
Vehicle / solvent:
Sterile water

VEHICLE CONTROL SUBSTANCE
The test substance was found to be soluble in sterile water, which was used as the vehicle control.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: acridine, daunomycin, 2-aminoanthracene
Remarks:
POSITIVE CONTROL SUBSTANCES
The performance of this test was evaluated with positive controls for each tester strain used, with and without metabolic activation (S9). Appropriate dilutions were prepared using the solvents listed below prior to testing.
Details on test system and experimental conditions:
Test System Identification
Each of the S. typhimurium and E. coli strains received for use on this study was accompanied by documentation that includes lot number, preparation and expiration dates, and confirmation of phenotype and response to specific mutagens. The following bacterial strains were purchased from Molecular Toxicology, Inc.:
Strain Characteristics Mutations Detected Lot Number Expiration Date
ST TA98 his; rfa; uvrB; R-factor Frameshift 5395D Aug 14, 2021
ST TA 100 his; rfa; uvrB; R-factor Base-pair substitution 5371D May 30, 2021
ST TA1535 his; rfa; uvrB Base-pair substitution 5360D Apr 03, 2021
ST TAI 537 his; rfa; uvrB Frameshift 5350D Mar 20, 2021
EC WP2 uvrA trp; uvrA Base-pair substitution 535 ID Mar 20, 2021

Legend:
his: histidine required as a growth factor
rfa: deep rough mutation involves loss of a major component of the cell coat increasing permeability to larger molecules; this deletion also involves the gene coding for biotin synthesis
uvr A/B: deletion of DNA nucleotide excision repair system
R-factor: contains the pKMIOI plasmid which increases sensitivity by enhancing error-prone DNA repair systems
trp: tryptophan required as a growth factor

Growth Media and Plates
Overlay agar (supplemented with biotin and limited amounts of histidine and tryptophan) and minimal glucose agar plates were purchased from Molecular Toxicology. Inc.

Bacteria (Test Systems)
Fresh bacterial suspension cultures in nutrient broth were prepared so that they were in the late exponential phase of growth at the time of use (approximately 1 x 10^9 bacteria/mL). Bacterial growth was evaluated by spectrophotometric optical density measurement.

Main Test
The initial test followed the plate incorporation method, in which the following materials were mixed and poured over the surface of a minimal agar plate:
• 100 uL of the prepared test substance solutions, negative (vehicle) control, or prepared
positive control substance
• 500 uL S9 mix or substitution buffer
• 100 uL bacteria suspension (ST or EC)
• 2000 uL overlay agar maintained at approximately 45 °C
Plates were prepared in triplicate at each experimental point and uniquely identified. After pouring, plates were placed on a level surface until the agar gelled then inverted and incubated at approximately 37°C until growth was adequate for enumeration (approximately 65 hours). Appropriate sterility control check plates (treated with critical components in the absence of bacteria) were included as a standard procedural check.

Confirmatory Test
The confirmatory test employed the pre-incubation modification of the plate incorporation test. The test or control substances, bacteria suspension, and S9/substitution buffer were incubated under agitation for approximately 30 minutes at approximately 37°C prior to mixing with the overlay agar and pouring onto the minimal agar plates before proceeding as described for the initial test. The study design for the confirmatory test, including strains, dose levels etc. was as described above for the initial (main) test.

Control of Bias
General procedures associated with the balanced design and conduct of this study were employed to control bias.

Results
After incubation, the number of colonies per plate was counted manually and/or with the aid of a plate counter (Colony Plate Reader: Model Colony-Doe-It). The mean and standard deviation were calculated for each set of triplicate plates.

Criteria for Validity
The background lawn for vehicle control plates should appear normal (i.e., slightly hazy with abundant microscopic non-revertant bacterial colonies). The mean revertant colony counts for each strain treated with the vehicle should lie close to or within the expected range taking into account the laboratory historical control range and/or published values (Mortelmans & Zeiger, 2000; Gatehouse, 2012). The positive controls (with SQ where required) should produce substantial increases in revertant colony numbers with the appropriate bacterial strain as specified in the Evaluation of Mutagenicity Section below.
In the case where part of the study is invalid based on these criteria (e.g.. the positive control does not induce an appropriate response with an individual strain or generally poor growth of the background lawn with that strain), detailed results for that part of the study will not be reported and the affected part of the study would normally be subjected to an automatic repeat at the discretion of the Study Director in consultation with the Sponsors described in an amendment, if appropriate.
Rationale for test conditions:
Justification for the Selection of the Test System
The referenced guidelines (Section 10) accept the combination of S. typhiniurium (TA98, TA 100, TAI 535, and TM537) and E.coli (WP2 uvrA) strains selected for use in this study.
Evaluation criteria:
Evaluation of Toxicity
Toxic effects of the test substance are indicated by the partial or complete absence of a background lawn of non-revertant bacteria (colony counts, if any, should not be reported) or a substantial dose-related reduction in revertant colony counts compared with lower dose levels and concurrent vehicle control taking into account the laboratory historical control range. Where precipitation obscures observations on the condition of the background lawn, the lawn can be considered normal and intact if the revertant colony counts are within the expected range based on results for lower dose levels and historical control counts for that strain.

Evaluation of Mutagenicity
For each experimental point, the Mutation Factor (MF) was calculated by dividing the mean revertant colony count by the mean revertant colony count for the corresponding concurrent vehicle control group. The mutagenic activity of the test item was assessed by applying the following criteria:
The results were considered positive (i.e., indicative of mutagenic potential) if:
• The results for the test item showed a substantial increase in revertant colony counts, i.e., response ME>2 for strains TA98, TAIOO, and WP2 uvrA or MF>3 for strains TA1535 and TA 1537, with mean value(s) outside the laboratory historical control range. Otherwise, results were considered negative.
• The above increase must be dose related and/or reproducible. i.e., increases must be obtained at more than one experimental point (at least one strain, more than one dose level, more than one occasion or with different methodologies).

If the second criterion is not met, the results may be classified as equivocal, and further testing may be appropriate.

A test substance that produces neither a concentration related increase in the number of revertant colonies nor a reproducible substantial increase in revertant colonies is considered to be non-mutagenic in this test system.
Statistics:
STATISTICAL ANALYSIS
Product Safety Labs calculated means and standard deviations for all quantitative data collected.

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS
Revertant colony counts for each strain are presented in Tables 1-5. Historical Control Data is presented in Appendix A. The Sterilization report is presented in Appendix B.

The mean revertant colony counts for each strain treated with the vehicle were close to or within the expected range, considering the laboratory historical control range and/or published values (Mortelmans & Zeiger, 2000; Gatehouse, 2012). The positive control substances caused the expected substantial increases in revertant colony counts in both the absence and presence of S9 in each phase of the test confirming the sensitivity of the test and the activity of the S9 mix. Therefore, each phase of the test is considered valid.

No signs of precipitation or toxicity were noted in any of the strains. Individual plate contamination which did not obscure counts was observed for TA98 at 158 pig/plate without S9 and for TAIOO at 5 pig/plate with S9 in the plate incorporation method. Contamination did not impact mutagenic evaluation.

For all strains, at least eight nontoxic dose levels were evaluated; therefore bacterial mutagenicity was adequately assessed.

There was no concentration-related or substantial test substance related increases in the number of revertant colonies observed with strains TA535, TA1537, TA98 , TAIOO or E.coli WP2 uvrA in both the absence and presence of S9 using either the plate incorporation or the pre-incubation method.

Any other information on results incl. tables

References:


Ames, B. N., Durston, W. B., Lee, F. D., & Yamasaki, B. (1973). Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection. Proceedings of the National Academy of Sciences, Vol. 70 (No. 8), 2281-2285.


Gatehouse D (2012). Bacterial mutagenicity assays: test methods. Methods in Molecular Biology, Vol. 817: 2 1-34.


Maron, D. M., & Ames, B. N. (1983). Revised methods for the Salmonella mutagenicity test. Mutation Research, 113, 173-215.


Mortelmans, K., & Zeiger, B. (2000). The Ames Salmonella/microsome mutagenicity assay. Mutation Research, 455, 29-60.


U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention & National Institutes of Health. (2009). Biosafety in Microbiological and Biomedical Laboratories (5th ed). HI-IS Publication No. (CDC) 21-1112.

Applicant's summary and conclusion

Conclusions:
Based on these findings and on the evaluation system used, FBS Transit did not elicit evidence of
bacterial mutagenicity in the Ames assay.