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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A reliable chromosome aberration study with human lymphocytes exposed to single superphosphate showed no genotoxicity.

In addition, reliable in vitro studies with other substances from the phosphate group were present. An Ames test with triple superphosphate showed negative results in the presence and absence of metabolic activation. A reliable in vitro TK assay with ammonium dihydrogenorthophosphate also showed negative results in the presence and absence of metabolic activation. The read-across rationale can be found in the category approach document attached in the target record and in the appendix of the CSR.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 April 2001 -21 May 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
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 / 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 µg/plate
Confirmatory mutagenicity assay: 50, 150, 500, 1250, and 3125 μg/plate for Salmonella and 150, 500, 1250, 3125, and 5000 μg/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 articla 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.
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 Migrated to IUCLID6: 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, 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.
Key result
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 ug/plate and with 200 ul of solvent control/plate. Confirmatory mutagenicity assay: slight or moderate reduction in backgroundlawn at 125 uL vehicle controls and corresponding test article aliquots
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:
cytotoxicity
Remarks:
Confirmatory mutagenicity assay: slight or moderate reduction in backgroundlawn at 200 uL 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 ug/plate

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 (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

(μg/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

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, Granular Triple Super Phosphate (GTSP)
did not cause a positive response in the presence and absence of Aroclor-induced rat liver S9.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12-mar-2010 to 25-apr-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human peripheral blood
Details on mammalian cell type (if applicable):
- Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.

- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.

- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Without and with S9-mix, 3hr exposure; 24 hr fixation: 3, 10, 33, 100 and 333 µg/mL
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 3, 10, 33, 100, 333 and 1000 µg/mL
First cytogenetic test:
Without and with S9-mix, 3 h exposure time, 24 h fixation time: 33, 100 and 333 µg/mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 33, 100 and 1000 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 33, 100 and 1000 µg/mL
With S9-mix, 3 hr exposure; 48 hr fixation: 33, 100 and 333 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Single super phosphate (SSP) was suspended in dimethyl sulfoxide. The stock solution was blended and treated with ultrasonic waves to obtain a homogeneous suspension. The test substance was vortexed and immediately the necessary quantity was taken and added to the culture medium.
- Justification for choice of solvent/vehicle: A homogeneous suspension could be obtained in DMSO and DMSO has been accepted and approved by authorities and international guidelines
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9; 0.5 µg/ml for a 3 h exposure period, 0.2 µg/ml for a 24 h exposure period and 0.1 µg/ml for a 48 h exposure period (in Hank's Balanced Salt Solution)
Positive control substance:
cyclophosphamide
Remarks:
with S9; 10 µg/mL in Hank's Balanced Salt Solution
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
Statistics:
The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.
Key result
Species / strain:
lymphocytes: human peripheral blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 333 µg/ml and above

RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to and including the highest precipitating tested dose

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Precipitation was seen at the dose levels selected for scoring.

No effects of Single super phosphate (SSP) on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Single super phosphate (SSP) does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Overview table of the results

Treatment Fixation Metabolic  Concentrations Cytotoxic Clastogenic
hrs hrs activation µg/mL    
3 24 no 33, 100 and 333 No No
3 24 yes 33, 100 and 333 No No
24 24 no 33, 100 and 1000 Yes No
48 48 no 33, 100 and 1000 Yes No
3 48 yes 33, 100 and 333 Yes No
Conclusions:
The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Single super phosphate (SSP) did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments.

Finally, it is concluded that this test is valid and that Single super phosphate (SSP) is not clastogenic in human lymphocytes under the experimental conditions described in the report.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
-RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Without and with S9-mix, 3 hours treatment: 3, 10, 33, 100 and 333 µg/mL
Without S9-mix, 24 hours treatment: 3, 10, 33, 100 and 333 µg/ml and 0.003, 0.01, 0.03, 0.1, 0.3, 1 and 3 µg/mL
Experiment 1:
Without S9-mix, 3 hours treatment: 0.003, 0.03, 0.1, 0.25, 0.5, 1, 1.4 and 2 µg/mL
With S9-mix, 3 hours treatment: 0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 12 µg/mL
Experiment 2
Without S9-mix, 24 hours treatment: 0.01, 0.03, 0.1, 0.25, 0.5, 1, 1.4 and 1.8 µg/mL
With S9-mix, 3 hours treatment: 0.01, 0.1, 1, 10, 12, 14, 16 and 17 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: RPMI 1640 medium
- Justification for choice of solvent/vehicle:Test compound was soluble in RPMI 1640 medium and RPMI 1640 medium has been accepted and approved by authorities and international guidelines
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9;15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period
Positive control substance:
cyclophosphamide
Remarks:
with S9; 7.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplo cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)
Evaluation criteria:
The global evaluation factor (GEF) has been defined by the IWTG as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more then MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: No precipitation was observed up to and including the top dose of 850 µg/mL (= 0.01 M)

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 3 µg/mL in the absence of S9, 3 hours treatment; at dose levels of 33 µg/mL in the presence of S9, 3 hours treatment; at dose levels of 1 µg/mL in the absence of S9, 24 hours treatment

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 79 and 83% compared to the total growth of the solvent controls after the 3 and 24 hours treatment period, respectively.

In the presence of S9-mix, no toxicity was observed up to and including the highest tested dose level in both experiments.
Conclusions:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

Mutation frequencies in cultures treated with positive control chemicals were increased by 11- and 16-fold for MMS in the absence of S9-mix, and by 19- and 11-fold for CP in the presence of
S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, Ammonium dihydrogenorthophosphate did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.

In the presence of S9-mix, Ammonium dihydrogenorthophosphate did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation.

It is concluded that Ammonium dihydrogenorthophosphate is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In accordance with column 2 of REACH Annex VII-X, no in vivo genotoxicity study (required in section 7.4) seems to be necessary, as the in vitro studies show no genotoxicity for the substance.

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

With single superphosphate only a chromosome aberration study is present. A reliable Ames test and TK-assay is not available with the substance itself.

In an Ames test with triple superphosphate according to OECD 471 guideline, Salmonella Typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and E. Coli WP2 uvr A showed no genotoxicity with and without metabolic activation, showing cytotoxicity at the highest concentrations tested. In an vitro chromosome aberration test with CHO cells performed according to OECD 473 guideline, also no genotoxicity was seen with and without metabolic activation when exposed to single superphosphate. Precipitation was observed. With ammonium dihydrogenorthophosphate a reliable TK-assay was present. In a Thymidine kinase (TK) assay in L5178Y mouse lymphoma cells performed according to OECD 476 and EC B.17 guidelines, the substance did not induce a significant increase in the mutation frequency. Based on these negative results for genotoxicity in in vitro studies, no in vivo studies are necessary.


Justification for selection of genetic toxicity endpoint
An Ames test with the read-across substance triple superphosphate and a mouse lymphoma study with the read-across substance Ammonium dihydrogenorthophosphate are available, showing no adverse effects. Since the study was performed with a substance analogue and the data are read across, the Klimisch score is 2. A reliable Chromosome aberration study with the substance is available, showing no adverse effects.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data, single superphosphate does not have to be classified according to the CLP Regulation for genetic toxicity.