Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 April 2001 -17 May 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Ninth Addendum to the OECD Guidelines for the Testing of Chemicals, published by OECD, Paris, February 1998.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals (1997).
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Diammonium phosphate (DAP)
IUPAC Name:
Diammonium phosphate (DAP)
Constituent 2
Chemical structure
Reference substance name:
Diammonium hydrogenorthophosphate
EC Number:
231-987-8
EC Name:
Diammonium hydrogenorthophosphate
Cas Number:
7783-28-0
Molecular formula:
H3N.1/2H3O4P
IUPAC Name:
diammonium hydrogen phosphate
Test material form:
other: particles
Details on test material:
- Name of test material (as cited in study report): Diammonium Phosphate (DAP)
- Substance type: brown discrete particles
- Physical state: solid

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: 2.5, 7.5, 25, 75, 200, 600, 1800 and 5000 ug per plate.
Confirmatory mutagenicity assay: 50, 150,500, 1500 and 5000 ug per plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile distilled water (CAS# 7732-18-5), obtained from Life Technologies, Inc.
- Justification for choice of solvent/vehicle: Based on compatibility with the target cells and workability of the test article in water.
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 ug/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: 1.0 ug/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: 1.0 ug/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: 75 ug/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: 1000 ug/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, TA1OO 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
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Precipitation conc: Initial Toxicity Mutation Assay: 1,800 μg/plate
Confirmatory Mutagenicity Assay: 5,000 µg/plate

Any other information on results incl. tables

Initial Toxicity-Mutation Assay

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

Test substance concentration

(μg/plate)

Number of colonies/platea)

Base-pair substitution type

Frame shift type

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9 mix

Solvent control

130

18

13

12

7

2.5

101

12

12

10

4

7.5

118

16

14

14

4

25

103

17

11

19

6

75

102

16

16

14

5

200

128

14

11

13

7

600

91

14

10

16

6

1800

100 *

14 *

12 *

12 *

5 *

5000

83 *

16 *

10 *

12 *

5 *

+S9 mix

Solvent control

145

21

14

15

9

2.5

119

9

18

16

9

7.5

124

11

14

18

6

25

117

12

17

19

7

75

99

11

14

13

6

200

110

14

13

21

6

600

92

12

17

15

7

1800

90 *

13 *

19 *

17 *

9 *

5000

95 *

15 *

9 *

13 *

6 *

Positive control without S9 mix

Name

NaN3

NaN3

MMS

2-NF

9-AA

Number of colonies/plate

396

176

94

97

343

Positive control with S9 mix

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Number of colonies/plate

486

87

167

459

70

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 = water

Confirmatory Mutagenicity Assay

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

Test substance concentration

(μg/plate)

Number of colonies/platea)

Base-pair substitution type

Frame shift type

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9 mix

Solvent control

119

20

16

12

6

50

101

17

12

12

5

150

136

17

15

9

5

500

105

17

15

10

9

1500

121

20

13

11

7

5000

125 *

19 *

10 *

11 *

5 *

+S9 mix

Solvent control

148

13

19

20

8

50

144

15

17

17

7

150

125

15

12

18

8

500

117

17

13

20

8

1500

128

14

16

16

7

5000

117 *

15 *

10 *

21 *

5 *

Positive control without S9 mix

Name

NaN3

NaN3

MMS

2-NF

9-AA

Number of colonies/plate

475

244

100

113

475

Positive control with S9 mix

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Number of colonies/plate

1094

143

239

977

97

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 = water

Applicant's summary and conclusion

Conclusions:
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, Diammonium Phosphate (DAP) did not cause a positive response in the presence and absence of Aroclor-induced rat liver S9.