Bacterial Reverse Mutation Test

KIWITA
Jan 15
4 min read

Updated: Jan 18

Date: October 21, 2008

Bacterial Reverse Mutation Test of

Water-Soluble Silica UMO

Test Period : September 8, 2008 – October 21, 2008

Test Requester : APA Corporation

Background of the Study

At the request of APA Corporation, the safety of water-soluble silica UMO was evaluated using a bacterial reverse mutation test.

Test Results

Both the preliminary test and the main test yielded negative results. It was therefore inferred that the test substance does not possess toxicity that induces gene mutations.Details are provided in the attached Final Report.

GLP Statement

Study Title:Bacterial Reverse Mutation Test of Water-Soluble Silica UMO

This study was conducted in accordance with Ministry of Health Ordinance No. 21, “Standards for the Conduct of Non-Clinical Studies on the Safety of Pharmaceuticals” (March 26, 1997; partially revised by Ministry of Health, Labour and Welfare Ordinance No. 114 dated June 13, 2008).

However, the measurements of the characteristics and stability of the test substance, as well as the stability and homogeneity of mixtures of the test substance and vehicle described in Article 13, were not conducted.

Date: October 21, 2008

Final Report

Bacterial Reverse Mutation Test of Water-Soluble Silica UMO

Test Period: September 3, 2008 – October 21, 2008

Date: October 21, 2008

1. Summary

This study was conducted under commission from the Institute of Applied Biology Co., Ltd. The mutagenic potential of water-soluble silica UMO was evaluated using bacterial test systems.

The following strains were used:

Base-pair substitution detection:Salmonella typhimurium TA100, TA1535Escherichia coli WP2uvrA
Frameshift mutation detection:Salmonella typhimurium TA98, TA1537

The pre-incubation method was employed. Tests were conducted both with and without metabolic activation (S9 mix).

Dose-range finding test:7 doses from 7 to 5000 μg/plate (common ratio 3)
Main test:6 doses from 156.3 to 5000 μg/plate (common ratio 2)

Results showed no reproducible, dose-dependent increases of revertant colonies to twice or more than those of the negative control in any strain.

Conclusion: Under the conditions of this study, water-soluble silica UMO was judged to have no mutagenic activity in any of the five bacterial strains tested.

2. Objective

To evaluate the gene mutation-inducing potential of water-soluble silica UMO using bacterial systems.

3. Study Personnel

Preparation of test substance solutions: Yuko Yamaguchi
Bacterial reverse mutation test: Masashi Imamura

4. Study Schedule

Study start: September 3, 2008

Dose-range finding test

Pre-culture: September 8, 2008
Test substance addition: September 9, 2008
Plate observation: September 11, 2008

Main test

Pre-culture: September 23, 2008
Test substance addition: September 24, 2008
Plate observation: September 26, 2008
Study end: October 21, 2008

5. Standards and Guidelines

GLP

Ministry of Health Ordinance No. 21 (March 26, 1997; revised June 13, 2008).Measurements of test substance characteristics, stability, and mixture homogeneity were not conducted.

Guideline

Guidelines for Genotoxicity Testing of Pharmaceuticals (Notification No. 1604, November 1, 1999).

6. Materials and Methods

6.1 Test Substance

Name: Water-Soluble Silica UMO (UMO)
Supplier: Test sponsor
Lot No.: 20080818 (Manufactured August 18, 2008)
Description: Mineral composite beverage containing water-soluble silica
Appearance: Clear (occasional mineral precipitation), alkaline
Storage: Light-protected, airtight, room temperature (1–30°C)
Handling: Protective clothing, mask, and gloves
Disposal: Returned to sponsor

6.2 Control Substances

6.2.1 Negative Control

Water for Injection (JP)
Manufacturer: Otsuka Pharmaceutical Factory, Inc.

6.2.2 Positive Controls

Sodium azide
AF-2 (2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide)
2-Aminoanthracene
9-Aminoacridine hydrochloride

Prepared and stored according to standard procedures. Selected based on genotoxicity testing guidelines.

6.3 Preparation of Test Solutions

Test solutions were freshly prepared on the day of use and diluted with water for injection to the designated concentrations. Solutions were homogenized using a vortex mixer.

6.4 Bacterial Strains

S. typhimurium TA100, TA1535, TA98, TA1537
E. coli WP2uvrA

Obtained from the National Institute of Health Sciences and NITE.Stored frozen at ≤ –80°C.

6.5 Media

Nutrient broth for pre-culture
Minimal glucose agar plates
Soft agar (top agar) supplemented with histidine/biotin or tryptophan

6.6 Metabolic Activation System (S9 Mix)

Prepared from liver homogenates of phenobarbital/benzoflavone-treated male Sprague-Dawley rats. Composition complied with standard Ames test protocols.

6.7 Test Procedures

Pre-culture: Bacteria grown under shaking at 37°C
Reverse mutation test: Pre-incubation method, with and without S9 mix
Sterility test: Plate method

6.8 Dose Selection

Dose-range finding: 7–5000 μg/plate
Main test: 156.3–5000 μg/plate

6.9 Observations

Bacterial growth inhibition
Test substance precipitation
Revertant colony counts

6.10 Statistical Analysis

No statistical analysis was performed.

6.11 Evaluation Criteria

A result was considered positive if revertant colonies increased to twice or more than the negative control with dose dependency and reproducibility.Otherwise, results were judged negative.

7. Results

7.1 Dose-Range Finding Test

No precipitation or growth inhibition observed. No increase exceeding twice the negative control.

7.2 Main Test

Results consistent with the preliminary test. All strains showed negative responses.

7.3 Sterility Test

No microbial contamination detected.

8. Discussion and Conclusion

All acceptance criteria were met. No mutagenic activity was observed under any test condition.

Conclusion:Water-soluble silica UMO does not induce gene mutations in the bacterial reverse mutation test under the conditions of this study.

9. References

Internal laboratory data
Miyake et al. Genotoxicity Testing Q&A for Pharmaceuticals. Scientist Press, 2000.

Note: This paper is translated from the following URL. The content is provided for reference on the scientific research of the raw material only. Whether APA raw materials are used or not, we hope this research will help increase understanding and awareness of body minerals.