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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:
experimental study
Adequacy of study:
key study
Study period:
2009-05-27 till 2009-07-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to the OECD guideline in compliance to GLP standards

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material: Sodium hypophosphite; tested in the form of monohydrate as the anhydrous form (CAS 7681-53-0) is highly hygroscopic and difficult to handle without specific precautions.
- Physical state: white crystalline solid
- Supplier: Rhodia UK-Oldbury
- Batch number: 90113D
- Expiry date: June 2010
- Storage condition of test material: room temperature, stored in a tightly closed container, opening of the container restricted to the minimum needs
- Purity: 99.5%
- Impurities (identity and concentrations):
Phosphite (Na2HPO3): 0.14%
- Certificate of analysis:
Analysis number: ITC/09/01/04
Analysis date: 2009-01-27

Method

Species / strain
Species / strain / cell type:
other: S.typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (liver post mitochondrial fraction of rats induced with Aroclor 1254)
Test concentrations with justification for top dose:
- First mutagenicity experiment with and without S9 mix: 312.5, 625, 1250, 2500 and 5000 µg/plate
- Second mutagenicity experiment with and without S9 mix: 625, 1250, 2500, 3750 and 5000 µg/plate
Vehicle / solvent:
- Vehicle used: water (for injections)
- Justification for choice of solvent/vehicle: highly soluble in water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: see table 1
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- In agar (plate incorporation): first mutagenicity test and second mutagenicity test without S9 mix
- Preincubation (60 minutes at 37°C ) only in second mutagenicity test with S9 mix
- Exposure duration: 48h to 72h

SELECTION AGENT (mutation assays): agar containing traces of histidine and biotin, maintained at 45°C

NUMBER OF REPLICATIONS: two independent mutagenicity experiments each using three plates/dose-level

DETERMINATION OF CYTOTOXICITY
- Method: the evaluation of the toxicity was based on the decrease in the number of revertant colonies and/or thinning of the bacterial lawn.
Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result.
Reference to historical data or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.
Statistics:
No statistical analysis performed

Results and discussion

Test results
Species / strain:
other: S.typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Tested up to limit concentrations recommended by the test guideline
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None
- Effects of osmolality: None
- Evaporation from medium: None
- Water solubility: freely soluble at 50 mg/mL
- Precipitation: No presipitate observed
- Other confounding effects: None
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

- The number of revertants for the vehicle and positive controls were as specified in the acceptance criteria. The study was therefore considered as valid.

 

- Since the test item was freely soluble and non-toxic in the preliminary test, the highest dose-level was 5000 µg/plate, according to the criteria specified in the international guidelines.

 

- No precipitate was observed in the petri plates when scoring the revertants at any of the tested dose-levels.

 

- No toxicity was noted at any dose-level in any strain.

 

- A noteworthy increase in the number of revertants was noted in the TA 1537 strain (up to 3.1-fold the vehicle control value) in the second experiment without S9 mix. This increase exceeded the threshold of 3-fold the vehicle control value, but it was not reproducible since it was not observed in the first experiment performed under the same experimental conditions. Moreover, the corresponding mean revertant colony counts remained within the historical data range for the vehicle control (11 versus 3-13 for the historical data). Consequently, this increase was not considered as biologically relevant.

 

- A slight increase in the number of revertants was noted in the TA 100 strain (up to 1.8-fold the vehicle control value) in the second experiment with S9 mix (preincubation method). Since this increase did not reach the threshold of 2-fold the vehicle control value and was neither observed in the first experiment (direct plate incorporation method) nor clearly dose-related, it was not considered as biologically relevant.

 

- The test item did not induce any other noteworthy increase in the number of revertants, in any of the other strains, either with or without S9 mix,.

- Table: Detailed results for the two mutagenicity test performed with five bacterial strains for five concentrations of the test substance sodium hypophosphite as well as the vehicle and positive control.

Strain Compound First mutagenicity test Second mutagenicity test
Dose level (µg/plate) S9 mix Revertant colony count Revertant colony count Dose level (µg/plate) S9 mix Revertant colony count Revertant colony count
Mean SD Mean SD
TA 1535 Water for injection without 13 5 without 29 8
Sodium hypophosphite 312.5 without 15 2 625 without 26 8
Sodium hypophosphite 625 without 15 5 1250 without 37 3
Sodium hypophosphite 1250 without 16 11 2500 without 30 8
Sodium hypophosphite 2500 without 13 6 3750 without 30 3
Sodium hypophosphite 5000 without 15 8 5000 without 33 14
Sodium azide 1 without 768 54 1 without 672 15
Water for injection with 16 5 with 25 10
Sodium hypophosphite 312.5 with 12 2 625 with 19 3
Sodium hypophosphite 625 with 14 4 1250 with 15 2
Sodium hypophosphite 1250 with 16 8 2500 with 30 11
Sodium hypophosphite 2500 with 23 6 3750 with 20 6
Sodium hypophosphite 5000 with 13 1 5000 with 21 7
2-Anthramine 2 with 184 22 2 with 87 7
TA 1537 Water for injection without 5 1 without 4 3
Sodium hypophosphite 312.5 without 9 4 625 without 6 4
Sodium hypophosphite 625 without 13 2 1250 without 7 1
Sodium hypophosphite 1250 without 7 1 2500 without 8 2
Sodium hypophosphite 2500 without 12 4 3750 without 8 3
Sodium hypophosphite 5000 without 8 4 5000 without 11 1
9-Aminoacridine 50 without 194 15 50 without 749 137
Water for injection with 12 7 with 10 1
Sodium hypophosphite 312.5 with 8 2 625 with 14 7
Sodium hypophosphite 625 with 5 1 1250 with 15 7
Sodium hypophosphite 1250 with 13 8 2500 with 12 6
Sodium hypophosphite 2500 with 8 9 3750 with 7 1
Sodium hypophosphite 5000 with 21 6 5000 with 10 4
2-Anthramine 2 with 111 6 2 with 56 6
TA 98 Water for injection without 30 6 without 26 4
Sodium hypophosphite 312.5 without 19 6 625 without 27 4
Sodium hypophosphite 625 without 22 3 1250 without 20 6
Sodium hypophosphite 1250 without 24 20 2500 without 27 4
Sodium hypophosphite 2500 without 26 3 3750 without 29 3
Sodium hypophosphite 5000 without 30 1 5000 without 21 4
2-Nitrofluorene 0.5 without 161 5 0.5 without 165 19
Water for injection with 35 5 with 25 8
Sodium hypophosphite 312.5 with 26 6 625 with 35 9
Sodium hypophosphite 625 with 39 5 1250 with 37 3
Sodium hypophosphite 1250 with 41 1 2500 with 39 4
Sodium hypophosphite 2500 with 37 6 3750 with 30 15
Sodium hypophosphite 5000 with 40 6 5000 with 32 8
2-Anthramine 2 with 741 38 2 with 872 148
TA 100 Water for injection without 124 8 without 136 11
Sodium hypophosphite 312.5 without 149 15 625 without 153 11
Sodium hypophosphite 625 without 148 8 1250 without 150 37
Sodium hypophosphite 1250 without 154 15 2500 without 156 20
Sodium hypophosphite 2500 without 155 13 3750 without 160 39
Sodium hypophosphite 5000 without 145 14 5000 without 149 18
Sodium azide 1 without 698 110 1 without 837 141
Water for injection with 147 8 with 92 9
Sodium hypophosphite 312.5 with 139 14 625 with 101 27
Sodium hypophosphite 625 with 145 36 1250 with 134 21
Sodium hypophosphite 1250 with 162 20 2500 with 135 30
Sodium hypophosphite 2500 with 163 25 3750 with 131 8
Sodium hypophosphite 5000 with 169 9 5000 with 167 17
Benzo(a)pyrene 5 with 353 16 5 with 563 72
TA 102 Water for injection without 372 18 without 374 39
Sodium hypophosphite 312.5 without 353 26 625 without 393 26
Sodium hypophosphite 625 without 347 32 1250 without 418 11
Sodium hypophosphite 1250 without 428 41 2500 without 451 21
Sodium hypophosphite 2500 without 386 20 3750 without 340 8
Sodium hypophosphite 5000 without 397 39 5000 without 389 18
Mitomycin C 0.5 without 3041 95 0.5 without 2081 158
Water for injection with 582 40 with 557 26
Sodium hypophosphite 312.5 with 566 77 625 with 469 29
Sodium hypophosphite 625 with 458 15 1250 with 528 105
Sodium hypophosphite 1250 with 470 24 2500 with 475 46
Sodium hypophosphite 2500 with 512 70 3750 with 548 95
Sodium hypophosphite 5000 with 586 31 5000 with 558 106
2-Anthramine 10 with 2775 29 10 with 1941 136

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under these experimental conditions, no noteworthy increase in the number of revertants was observed towards all the strains used, both with and without S9 mix. Sodium hypophosphite did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.
Executive summary:

The potential of sodium hypophosphite to induce reverse mutation in bacteria was assessed using five strains of Salmonella typhimurium according to the OECD guideline 471 and the EU Method B13/14. The study was conducted in compliance with the principles of Good Laboratory Practice on the monohydrated form of the test item as the anhydrous form is highly hygroscopic and difficult to handle without specific precautions.

A preliminary toxicity test was performed to define the dose-levels of sodium hypophosphite to be used for the mutagenicity study. The test item was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C).

 

The five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were exposed to the following dose-levels of sodium hypophosphite (three plates/dose-level):

- 312.5, 625, 1250, 2500 and 5000 µg/plate, for the first mutagenicity experiment with and without S9 mix,

- 625, 1250, 2500, 3750 and 5000 µg/mL for the second mutagenicity experiment with and without S9 mix.

After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

No precipitate was observed in the petri plates when scoring the revertants at all dose-levels.

No toxicity was noted towards all the strains used, both with and without S9 mix.

The test item did not induce any significant increase in the number of revertants, both with or without S9 mix, in any of the five strains.

Under these experimental conditions sodium hypophosphite did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.