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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:
4th March 2013 - 28th March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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:
Test Item: SE7B Batch 2137-0 (CAS 1365345-64-7)
Stability: Stable
Purity: 100%

Method

Target gene:
Tester strain Gene affected
E. coli WP2 uvrA T/PE
S. typh. TA-97a his D 6610
S. typh. TA-1535 his G 46
S. typh. TA-98 his D 3052
S. typh. TA-100 his G 46
Species / strainopen allclose all
Species / strain / cell type:
other: TA1535, TA-97a, TA-98 and TA-100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
A cytotoxicity screen was conducted in the Salmonella typhimurium TA-1 00 tester strain using eight concentrations (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, and 5 µl/plate) of the test article, two plates per dose.

Based on the cytotoxicity results, five concentrations (0.05, 0.1, 0.5, 1 and 5 µl/plate) of the test article were tested in each of five bacterial tester strains (E. coli WP2 uvrA, and S. typhimurium strains TA-97a, TA-1535, TA-98, and TA-100).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Based on test article solubility information provided by Sponsor, the Study Director chose Acetone as the vehicle for the assay.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
All bacterial strains with exogenous metabolic activation (S9).
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
E. coli WP2 uvrA without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Acridine, 6-chloro-9-(3-((2-chloroethyl)amino) propyl)amino-2-methoxy, dihydrochloride
Remarks:
S. typh. TA-97a without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
S. typh. TA-100 and TA-1535, without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Daunomycin (DM)
Remarks:
S. typh. TA-98 without S9
Details on test system and experimental conditions:
Tester strains were dosed in triplicate with five concentrations of the test article. Six plates each were dosed with the Vehicle Control and the Positive Control specific for each tester strain.

Preparation of the Tester Strains:
Bacterial cultures were inoculated by the addition of a lyophilized disk of each tester strain to Oxoid No.2 nutrient broth (Molecular Toxicology, Inc. (Moltox) Boone, NC, cat. #26-555). Ampicillin was added to the nutrient broth to ensure the retention of R-factor plasmid in tester strains TA-97a, TA-98 and TA-100. The cultures were incubated at 37°C ±2°C with agitation. The cultures were used after they reached the late exponential growth phase as determined by absorbance readings at 600 nm.

Treatment of the Test System:
Top agar supplemented with appropriate amino acids were prepared, as 2 ml aliquots, and maintained at 45-50°C in sterile culture tubes. Dulbecco's Phosphate Buffered Saline (DPBS) was added to the tubes not undergoing S9 activation (i.e. without S9, or -S9) to maintain equal dosing volumes. 0.1 ml of bacteria was added to the top agar, followed by 0.1 ml of the test article, Vehicle Control or Positive Control. For the activation portion of the test, 0.5 ml of S9 mixture was added last. The contents were gently vortexed and overlaid onto minimal glucose agar plates. After the mixture had solidified, the plates were incubated at 37°C ±2°C for 48-72 hours. Plates that were not scored immediately following the incubation period were stored at 2-8°C until scoring.

Main Assay:
Five concentrations (0.05, 0.01, 0.5, 1 and 5 µl/plate) of the test article were tested in each of five bacterial tester strains (Escherichia coli WP2 uvrA, and Salmonella typhimurium strains TA-97a, TA-1535, TA-98, and TA-100). Two sets of culture plates were dosed per concentration (+S9 and No S9). A Vehicle Control and Positive Controls specific to each bacterial strain were treated in a similar manner as the test article concentrations. The plates were incubated at 37°C ±2°C for 48-72 hours.

Revertant Colony Count:
Counting of the revertants per plate was performed using an Alphalmagerm 2200 (Alpha Innotech Corporation, San Leandro, CA) fluorescence imager. Proper function of the imager was verified against a standard template (e.g. high (1000), medium (100) and low (10) counts) prior to each daily use. The number of revertants was recorded, along with observations of cytotoxicity. Routine examination (under a light microscope) of the bacterial background lawn was used to determine cytotoxicity of the test article. The plates were also examined visually for test article precipitate.

Independent Repeat Assay:
No positive response or dose-related increased response of the test article in any strain was found in the main assay, so an independent repeat assay was conducted with the same test conditions used in the main study.
Evaluation criteria:
Analysis of Data:
Plates were scored based on the number of revertant colony-forming units present per plate. The number of revertants of each test article plate were averaged and plotted versus concentration of the test article. The mean number of revertants of each dose was divided by the mean for the Vehicle Control value to obtain a ratio to vehicle. In evaluating the data, cytotoxicity of the test article as well as quality checks of the assay were taken into account.

In general, a 2-fold increase with or without metabolic activation is considered a positive response. Dose related increases approaching a 2-fold increase are deemed equivocal.

A negative result is determined by the absence of a dose-related increase in all five tester strains, again taking into account cytotoxicity of the test article as well as the quality checks of the assay.

Positive results from the bacterial reverse mutation test indicate that the substance induces point mutations by base substitutions or frame shifts in the genome of either Salmonella typhimurium and/or Escherichia coli. Negative results indicate that under the test conditions, the test substance is not mutagenic in the tested species.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: TA1535, TA-97a, TA-98 and 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 applicable
True negative controls validity:
not specified
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
There was no diminution and clearing of the background lawn observed at any dose level, indicating that the test article was not cytotoxic to TA-100 at 0.001 to 5 µl/plate. The test article precipitated out in the plates at concentrations of 0.5 to 5 µl/plate. The Study Director chose 5 µl/plate as the top test article concentration for the main test.

Main Assay:
The assay was run in all five strains on triplicate plates. Positive and Vehicle Controls were run concurrently for all five strains, on six plates per strain. All plating was with and without exogenous metabolic activation, S9. No reduction or clearing of the bacterial background lawn was observed, indicating no or minimal cytotoxicity of the test article under test conditions. The test article precipitated out in the plates at 0.5 to 5 µl/plate. However, the precipitation did not interfere with the automatic counting of the plates. There was no significant increase or dose-dependent increase of the number of revertants in any tester strain treated with the test article in the presence or absence of S9. All Positive and Negative Control values were within acceptable ranges, and all criteria for a valid study were met.
Remarks on result:
other: all strains/cell types tested

Any other information on results incl. tables

Sterility Test: No contaminating microorganisms were detected in any of the reagents used in the assay. No contaminating microorganisms were detected in any of the reagents used in the assay. The sterility test passed the quality checks.

Vehicle Controls The spontaneous reversion rate, as represented by the mean colony forming units (CFU), for each strain of bacteria was measured and compared to in-house historical ranges. All Vehicle Controls passed the quality check.

Positive Controls: All Vehicle Controls passed the quality check.

Applicant's summary and conclusion

Conclusions:
Under test conditions, test article CAS# 1365345-64-7 (SE7B Batch 2137-0), did not have mutagenicity potential in the Bacterial Reverse Mutation Test.
Executive summary:

Objective:

The purpose of this study is to evaluate the mutagenic potential of a test article based on the reversion of selective growth mutations in several strains of Salmonella typhimurium bacteria and in Escherichia coli WP2 uvrA bacteria, in the presence and absence of S9 activation. This protocol is based on OECD Guideline for Testing of Chemicals: No. 471 - Bacterial Reverse Mutation Test and U.S. EPA Health Effects Test Guidelines OPPTS 870.5100 - Bacterial Reverse Mutation Test.

Method Synopsis:

Based on test article solubility information provided by Sponsor, the Study Director chose Acetone as the vehicle for the assay. A cytotoxicity screen was conducted in the Salmonella typhimurium TA-1 00 tester strain using eight concentrations (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, and 5 µl/plate) of the test article, two plates per dose. The test article was combined with the bacteria and top agar in the presence and absence of a metabolic activation mixture (S9) and overlaid onto minimal glucose agar plates. An Acetone Vehicle Control was run concurrently, with and without S9. Based on the cytotoxicity results, five concentrations (0.05, 0.1, 0.5, 1 and 5 µl/plate) of the test article were tested in each of five bacterial tester strains (E. coli WP2 uvrA, and S. typhimurium strains TA-97a, TA-1535, TA-98, and TA-100). Vehicle Controls and Positive Controls specific to each bacterial strain were treated in a similar manner as the test article concentrations. The plates were incubated at 37°C ±2°C for 48-72 hours. Revertant colony growth was determined by counting the colonies per plate using an Alphalmagerm imaging system. The number of revertants of the test article treatment plates and Positive Control plates was divided by the number of revertants of the vehicle plates. In general, a positive result is determined by a 2-fold increase above the Vehicle Control. Due to a negative result in the main assay, an independent repeat assay (confirmatory test) was conducted in all five tester strains using test article concentrations of 0.05, 0.1, 0.5, 1 and 5 µl/plate.

Summary:

Test article CAS# 1365345-64-7 (SE7B Batch 2137-0), in the vehicle, Acetone, was tested in a Bacterial Reverse Mutation Assay. The test article did not show obvious cytotoxicity to tester strain TA-100 at a dose range of 0.001 to 5µl/plate, with or without S9. The test article at 0.05 to 5µl/plate, with or without S9, did not cause a significant increase or a dose-dependent increase of the number of revertants of any bacterial tester strain, indicating that the test article is negative for mutagenicity in the Bacterial Reverse Mutation Assay. As in the main test, in the independent repeat assay the test article at 0.05 to 5 µl/plate, with or without S9, did not cause a significant increase or a dose-dependent increase of the number of revertants of any bacterial tester strain, confirming that the test article is negative for mutagenicity in the Bacterial Reverse Mutation Assay.

Conclusion:

Under test conditions, test article CAS# 1365345-64-7 (SE7B Batch 2137-0), did not have mutagenicity potential in the Bacterial Reverse Mutation Test.