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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:
26 April 2001 -21 May 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well-documented, GLP-compliant, according to OECD 471 in Salmonella typhimurium and E. coli. The rationale to use data from individual constituents and components of the complex is explained in chapter 1 of the CSR and in the adjacent "read-across document".

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

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
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Granular Triple Super Phosphate (GTSP) 45.43% P2O5 equivalent
- Substance type: Brown discrete particles
- Physical state: solid
- Analytical purity: 100% (provided by sponsor)
- Lot/batch No.: June 8, 2000
- Expiration date of the lot/batch: June 8, 2001
- Stability under test conditions: not indicated
- Storage condition of test material: room temperature, protected from light and moisture

Method

Target gene:
Salmonella tester strain cultures: deep rough mutation (ifa) and the deletion in the uvrB gene.
Cultures of tester strains TA98 and TA100: presence of the pKM101 plasmid R-factor.
All WP2 uvrA cultures: deletion in the uvrA gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: TA98 + TA1537: reverted from histidine dependence to histidine independence by frameshift mutagens. TA1535: reverted by mutagens that cause basepair substitutions. TA100: reverted by mutagens that cause both frameshift and basepair substitution mutations
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: Specificity of the reversion mechanism in E. coli is sensitive to base-pair substitution mutations, rather than frameshift mutations
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9-mix from male Sprague-Dawley rats
Test concentrations with justification for top dose:
Initial toxicity-mutation assay: 125, 250, 375, 500, 625, 1250, 2500 and 5000 microg/plate
Confirmatory mutagenicity assay: 50, 150, 500, 1250, and 3125 microg/plate for Salmonella and 150, 500, 1250, 3125, and 5000 microg/plate for E. coli
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 1 N HCl (CAS 7647-01-0), obtained from Fischer Scientific. 1 N HCl was heated to 50 °C for approximately 30 minutes
prior to use in test article preparation, All dilutions were held at 50 °C during dosing.
- Justification for choice of solvent/vehicle: Based on compatibility with the target cells and workability of the test article.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene: 1.0 and 10 microg/plate
Remarks:
All tester stains with S9 activation mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA 98 without S9 activation mix

Migrated to IUCLID6: 1.0 microg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 100 and TA 1535 without S9 activation mix

Migrated to IUCLID6: 1.0 microg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA 1537 without S9 activation mix

Migrated to IUCLID6: 75 microg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA without S9 activation mix

Migrated to IUCLID6: 1000 microg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in top agar (plate incorporation)

DURATION
- Preincubation period: overnight (To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored)
- Exposure duration: approximately 48 to 72 hours at 37+/-2 °C

NUMBER OF REPLICATIONS:
Initial Toxicity-Mutation Assay in duplicate
Confirmatory Mutagenicity Assay in triplicate

DETERMINATION OF CYTOTOXICITY
The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope.

OTHER EXAMINATIONS:
- Other: Precipitate was evaluated by visual examination without magnification.

OTHER:
Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.
Evaluation criteria:
For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than three times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean vehicle control value.
Statistics:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated.
Since no positive responses were observed, no statistical analysis of the responses was performed.

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:
Inital toxicity-mutation assay: toxicity at 5000 micg/plate and with 200 micL of solvent control/plate. Confirmatory mutagenicity assay: slight or moderate reduction in background lawn at 125 micL vehicle controls and corresponding test article aliquots
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Confirmatory mutagenicity assay: slight or moderate reduction in background lawn at 200 microL vehicle controls and corresponding test article aliquots
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At 1250-5000 microg/plate
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Initial toxicity-mutation assay:

With (+) or Without (-) S9-mix

Test substance concentration

(microg/plate)

Number of colonies/platea)

Base-pair substitution type

Frame shift type

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9 mix

Solvent control (25 µl)

176

15

13

12

6

Solvent control (50 µl)

162

11

11

12

7

Solvent control (100 µl)

106

9

7

5

8

Solvent control (200 µl)

70

4

8

4

4

125

183

13

8

7

6

250

168

18

9

10

6

375

144

14

7

11

7

500

168

10

9

8

5

625

174

13

16

11

7

1250

154 *

11 *

9 *

13 *

8 *

2500

112 *

10 *

9 *

6 *

4 *

5000

87 *

7 *

10 *

6 *

5 *

+S9 mix

Solvent control (25 µl)

204

17

12

19

13

Solvent control (50 µl)

168

9

11

14

8

Solvent control (100 µl)

112

12

9

7

3

Solvent control (200 µl)

16

5

9

7

2

125

195

7

9

12

6

250

197

13

9

14

8

375

199

12

9

15

9

500

163

12

9

12

6

625

209

18

8

12

7

1250

168 *

15 *

9 *

16 *

10 *

2500

158 *

13 *

10 *

8 *

5 *

5000

120 *

6 *

11 *

7 *

4 *

Positive control without S9 mix

Name

NaN3

NaN3

MMS

2-NF

9-AA

Number of colonies/plate

588

196

96

184

891

Positive control with S9 mix

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Number of colonies/plate

1191

180

289

901

148

a) The average number of revertant colonies from two plates

*)Precipitate

NaN3= sodium azide; 2-NF= 2-nitrofluorene; 9-AA = 9-aminoacridine; MMS = methyl methanesulfonate; 2 -AA=2 -aminoanthracene

Solvent control = 1 N HCl

Confirmatory Mutagenicity assay:

With (+) or Without (-) S9-mix

Test substance concentration

(microg/plate)

Number of colonies/platea)

Base-pair substitution type

Frame shift type

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9 mix

Solvent control (50 µl)

125

13

12

15

4

Solvent control (125 µl)

63

11

5

8

2

Solvent control (200 µl)

 

 

7

 

 

50

124

12

 

10

4

150

117

10

12

14

5

500

100

13

11

10

4

1250

99 *

10 *

11

9 *

4 *

3125

38 *

9 *

9 *

9 *

3 *

5000

 

 

6 *

 

 

+S9 mix

Solvent control (50 µl)

138

11

10

14

6

Solvent control (125 µl)

61

7

9

10

5

Solvent control (200 µl)

 

 

8

 

 

50

127

8

 

15

6

150

131

11

14

13

5

500

123

10

11

11

5

1250

134 *

9 *

10

13 *

5 *

3125

95 *

10 *

11 *

8 *

3 *

5000

 

 

9 *

 

 

Positive control without S9 mix

Name

NaN3

NaN3

MMS

2-NF

9-AA

Number of colonies/plate

416

133

87

125

704

Positive control with S9 mix

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Number of colonies/plate

1410

65

87

583

192

a) The average number of revertant colonies from three plates

*)Precipitate

NaN3= sodium azide; 2-NF= 2-nitrofluorene; 9-AA = 9-aminoacridine; MMS = methyl methanesulfonate; 2 -AA=2 -aminoanthracene

Solvent control = 1 N HCl

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

All criteria for a valid study were met as described in the protocol.
The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, Granular Triple Super Phosphate (GTSP)
did not cause a positive response in the presence and absence of Aroclor-induced rat liver S9.
Executive summary:

The test item was assessed for its potential to induce gene mutations in the plate incorporation test according to OECD TG 471 in Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA100 as well as the Escherichia coli strain WP2 uvrA with and without metabolic activation (Aroclor 1254-induced rat liver S9-mix from male Sprague-Dawley rats) at concentrations of 125, 250, 375, 500, 625, 1250, 2500 and 5000 microg/plate in the initial toxicity-mutation assay and at concentrations of 50, 150, 500, 1250, and 3125 microg/plate for Salmonella and 150, 500, 1250, 3125, and 5000 microg/plate for E. coli in the confirmatory mutagenicity assay. All criteria for a valid study were met as described in the protocol. The results indicate that, under the conditions of this study, the test substance did not cause any positive response in the presence and absence of Aroclor-induced rat liver S9. The test item did not reveal any mutagenic activity. The appropriate reference mutagenes showed distinct positive mutagenic effects.