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Genetic toxicity: in vitro

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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:
The experimental phase of this study was performed between 26 September 2012 and 09 November 2012.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted to GLP and in compliance with agreed protocols, with no deviations from standard test guidelines and no methodological deficiencies. This study is conducted according to an appropriate guideline and under the conditions of GLP, the study is therefore considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement as a key study for Regulation (EC) No. 1907/2006 (REACH). The reliability has been assigned in accordance with 'practical guide 6: How to report read-across and categories' which states that the maximum reliability for a read-across study is 2. The study is considered to be adequate and reliable for the purpose of registration under REACH (Regulation (EC) No. 1907/2006. Read-across in accordance with Annex XI, Section 1.5 of Regulation (EC) No. 1907/2006 (REACH) is justified on the following basis: Manganese phosphates such as manganese hydrogen phosphate and manganese bis(dihydrogen phosphate) are soluble manganese-containing inorganic compounds. The toxicology of these materials is considered to be related to the presence of the Mn2+ ion (as phosphate itself is not considered to be toxic). As such it is scientifically justified to read-across to other manganese phosphates. When considering a testing strategy for manganese phosphates, tests have been performed on manganese hydrogen phosphate as that contributes the greater amount of Mn2+ on a %w/w basis (36.4% as compared to 22% in manganese bis(dihydrogen phosphate. These results will be directly read across to manganese bis(dihydrogen phosphate) as they are representative of a worst-case for manganese phosphates.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspection: 10 July 2012, date of signature: 30 November 2012
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Manganese hydrogen phosphate
EC Number:
257-147-0
EC Name:
Manganese hydrogen phosphate
Cas Number:
51349-94-1
IUPAC Name:
manganese(2+) hydrogen phosphate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Sponsor's identification : IP 35: Manganese hydrogen phosphate
Description : Pale pink powder
Purity : 98%
Batch number : MV 500
EC Number : 257-147-0
CAS Number : 51349-94-1
Date received : 06 September 2012
Expiry date : Not supplied
Storage conditions : Room temperature in the dark
The integrity of supplied data relating to the identity, purity and stability of the test item is the responsibility of the Sponsor. A Certificate of Analysis was provided by the sponsor.

Method

Target gene:
Histidine for Salmonella.
Tryptophan for E.Coli
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:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta­naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
main test:
Experiment one: 50, 150, 500, 1500 and 5000 μg/plate
Experiment two: 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water, dimethyl sulphoxide, acetone, dimethyl formamide and acetonitrile at 50 mg/ml and tetrahydrofuran at 200 mg/ml in solubility checks performed in-house. The test item formed the best doseable suspension in dimethyl sulphoxide, therefore, this solvent was selected as the vehicle. The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 15 minutes at 40°C on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity content (2%) of the test item. All formulations were used within four hours of preparation an were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate for TA100
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
3 μg/plate
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix

Migrated to IUCLID6: at 3 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate for TA100
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene at 1 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate for TA1535
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
5 μg/plate
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix

Migrated to IUCLID6: at 5µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate for TA1535
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene at 2 μg/plate
Remarks:
with S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate for WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
2 μg/plate
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix

Migrated to IUCLID6: at 2 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate for WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene at 10 µg/plate
Remarks:
with S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate for TA98
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
0.2 μg/plate
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 mix

Migrated to IUCLID6: at 0.2 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous muation rate for TA98
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
5 μg/plate
Positive control substance:
benzo(a)pyrene
Remarks:
with S9 mix

Migrated to IUCLID6: at 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate for TA1537
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
80 μg/plate
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix

Migrated to IUCLID6: at 80 µg/plate
Untreated negative controls:
yes
Remarks:
Sponateous muation rate for TA1537
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene at 2 µg/plate
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 48 -72 hrs


SELECTION AGENT (mutation assays): Not applicable.


NUMBER OF REPLICATIONS: Triplicate plating.


NUMBER OF CELLS EVALUATED: Not applicable.


DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.


OTHER EXAMINATIONS: None
Evaluation criteria:
Acceptance Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are presented in the General Study Plan, Section 4 (negative controls). Combined historical negative and solvent control ranges for 2010 and 2011 can be found in the study report (Appendix 2).
All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per ml.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation. The historical ranges of the positive controls for 2010 and 2011 are presented inthe study report in Appendix 2.
There should be a minimum of four non-toxic test item dose levels.
There should be no evidence of excessive contamination.

Evaluation Criteria
There are several criteria for determining a positive result. Any, one, or all of the
following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).
Statistics:
Standard deviation

Results and discussion

Test resultsopen allclose all
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
Remarks:
Tested up to maximum recommended dose of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Tested up to maximum recommended dose of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test material formed the best doseable suspension in dimethyl sulphoxide at 50 mg/ml in solubility checks performed in-house.
- Precipitation: A pale, fine precipitate was observed at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: None
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary Toxicity Test

The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-)

 

The numbers of revertant colonies for the toxicity assay were:

 

With (+) or

without (-)

S9-mix

Strain

Dose (μg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

91

108

88

99

104

94

105

94

89

C

92P

+

TA100

99

121

128

131

109

134

125

114

116

108

92P

-

Wp2uvrA

20

33

18

26

28

27

32

29

27

24

28P

+

Wp2uvrA

27

40

37

31

43

21

28

28

31

29

16P

P = Precipitate

C – Contaminated 

Mutation Test

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9-mix used in both experiments was shown to be sterile.

The culture density for each bacterial strain was also checked and considered acceptable. These data are not given in the report.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.

 

A history profile of vehicle/untreated and positive control values (reference items) for 2010 and 2011 are presented in Appendix 2.

 

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. A test item precipitate (fine and particulate in appearance) was noted at 5000 μg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

 

 

All tables and appendices are provided as attachments due to the fact that they are too large to transcribe into the available free text fields.

The test item, IP 35: Manganese hydrogen phosphate, was considered to be non-mutagenic under the conditions of this test.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.

This study is conducted according to an appropriate guideline and under the conditions of GLP, the study is therefore considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement as a key study for this endpoint.

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