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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline conform GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Purity: > 99.999%;
Supplier: Sigma-Aldrich, Inc.;
Lot No.: 09823ME

Method

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:
rat liver S9 mix
Test concentrations with justification for top dose:
156.3, 312.5, 625, 1,250 and 5,000 μg/plate
Vehicle / solvent:
Sterilized distilled water;
Lot No.: 400644;
Manufacturer: GIBCO 15230-147
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Sterilized distilled water
Positive controls:
yes
Positive control substance:
other: sodium azide 2-Nitrofluorene, 9-Aminoacridine, 2-Aminoanthracene, 2-Aminofluorene
Details on test system and experimental conditions:
Six concentrations of the test substance were tested in triplicate against each tester strain, using a plate incorporation method.1) Master plate was prepared from the frozen permanent.2) A colony was selected from the master plate and inoculated to 10 ml of 1.6 % Oxoid Nutrient Broth (pH 7.0~7.5) for 12~14 hr by shaking at 200 rpm.3) The test substance (or vehicle, or positive controls) 0.1 ml , 0.1 ml og bacterial cultres, and 0.5 ml of S9 mix (or sodium phosphate buffer, 0.2 M, pH 7.4) were added to a test tube and then, 2 ml of molten top agar containing trace histidine or tryptophan was added to each tube. The mixture was overlaid onto sterile plates of Vogel-Bonner Minimal glucose agar.4) After approximately 48 hr incubation at 37 °C the frequency of revertant colonies was assessed.5) In order to assess the sterility of the test substance and S9 mix, 0.1 ml of the test substance at a maximal concentration (or 0.5 ml of S9 mix) and 2 ml of molten top agar were overlaid onto a sterile minimal glucose agar plate.6) Three plates was used at each dose levels.7) The plate was selected randomly in order to avoid a confounding effects.8) The project number, the name of bacteria strain, the treatment dose and absence or presence of S9 mix were recorded on the bottom of plate.
Evaluation criteria:
Data were presented using tables and figures as individual plate counts, the mean number of revertant colonies per plate and standard deviation.The following several criteria were used to determine a positive result.1) The case that under the S9- or S9+ condition, the increase of reverse mutation colony of a strain in a dose-dependent manner, detection of repeated increase at doses more than one concentration, or having a significant linear relation.Biological relevance of the results was considerd.2) An increase in revertant colonies over two times in bacterial strains TA98, TA100 and WP2uvrA, and over three times in strains TA1535 and TA1537 than those in negative control group.Besides the above cases, it was judged negative. Statistical significancy-analysis was not carried out.
Statistics:
none

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5,000 μg/plate in TA100 strain with S9 mix
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5,000 μg/plate in TA100 strain with S9 mix
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table1: Summary of revertant colony numbers obtained per plate with/without S9 mix

S9 mix

(5 %)

Dose

(ug/plate)*

Number of revertant colonies/plate

Base replacement type

Frame shift type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

NC

165+/-6

15+/-3

24+/-1

17+/-2

9+/-2

156.3

173+/-2

20+/-2

25+/-5

19+/-4

6+/-2

312.5

163+/-10

15+/-2

19+/-6

21+/-5

5+/-3

625

153+/-6

17+/-6

20+/-5

16+/-3

7+/-1

1250

158+/-6

17+/-6

14+/-2

16+/-3

5+/-1

2500

165+/-10

15+/-1

20+/-6

17+/-3

9+/-3

5000

166+/-2

11+/-4

23+/-7

20+/-7

5+/-4

PC

479+/-15

520+/-82

122+/-8

174+/-29

771+/-21

+

NC

174+/-5

11+/-2

22+/-3

29+/-6

11+/-0

156.3

161+/-16

13+/-4

18+/-3

25+/-10

6+/-3

312.5

156+/-5

15+/-5

19+/-4

25+/-8

8+/-2

625

160+/-7

18+/-5

16+/-3

25+/-8

10+/-3

1250

159+/-8

16+/-5

28+/-8

30+/-3

9+/-1

2500

154+/-26

18+/-2

18+/-7

18+/-4

9+/-2

5000

1+/-1

1+/-1

23+/-4

0+/-0

1+/-1

PC

587+/-30

144+/-22

329+/-37

493+/-59

317+/-31

Data are presented as mean+/-SD (N=3)

NC: negative control (Sterilized distilled water, 100 ul/plate*)

PC: positive control

*ug/plate, ul/plate: micro g/plate, micro l/plate

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Diphosphorus pentaoxide is a hygroscopic solid which forms with moisture/water an aqueous solution of phosphorus oxyacids (primary hydrolysis) that are subject to further (secondary) hydrolysis to the end product phosphoric acid, H3PO4. Therefore, the genotoxicity of Diphosphorus pentaoxide is solely based on secondary effects of Phosphoric acid and an read across to this substance is justified.

Under the present test conditions Phosphoric acid was considered to be non-genotoxic.
Executive summary:

Introduction

This study was conducted to evaluate the mutagenic potential of the test substance, phosphoric acid using a bacterial system. This study was performed in accordance with the procedures described in the following internationally accepted guidelines and recommendations: OECD Guidelines for Testing of Chemicals (Jul. 21, 1997) No. 471[ Bacterial Reverse Mutation Test] and [Genotoxicity Test] described in the TCCA-Good Laboratory Practice Standards and Test Guideline (NIER Public Notice No. 2007-29) issued by Korean National Institute of Environmental Research.

Dose range-finding test

Dose range-finding test was performed using theSalmonella typhimuriumstrains TA98, TA100 and theEscherichia colistrain WP2uvrA in the presence and absence of a metabolic activation containing S9 fraction (rat liver postmitochondrial fraction). The test substance at 5000 ug/plate* was used as a maximal concentrations in the absence and presence of S9 mix, in the main mutation test, and diluted to six doses (156.3, 312.5, 625, 1,250 and 5,000 ug/plate) by a factor two.

Bacterial reverse mutation test

In the main and the confirmation test, five bacterial strains,Salmonella typhimuriumTA98, TA100, TA1535, TA1537 andEscherichia coliWP2uvrA were treated with the test substance with or without S9 mix using a plate incorporation method and 3 plates at each dose levels were used (triplicates). As a result of the main mutation test, the test substance at any concentrations of 156.3~5,000 ug/plate with or without S9 mix didnt induce the increase in the frequency of revertant colonies in a dose-dependent manner, which representing non-genotoxic.

Regardless of S9 mix and tester strains, the precipitation of the test substance was not observed on the agar plate treated at any doses. In the confirmation test, the test substance at same concentrations of the main mutation test with or without S9 mix didnt induce the increase in the frequency of revertant colonies dose-related. The precipitation of the test substance was not observed on the agar plate treated at any doses.

The frequency of revertant colonies for the negative control, sterilized distilled water was considered to be acceptable and all of the positive controls induced marked increases in the frequency of revertant colonies confirming the activity of S9 mix and the sensitivity of the bacterial strains.

Conclusion

The test substance, phosphoric acid was considered to be non-genotoxic under the present test conditions.

*ug/plate: micro g/plate