Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No reliable data is available with tricalcium bis(orthophsphate). Reliable data is available with the read across substance pentacalcium hydroxide tris(orthophosphate).

In vitro (RA 12167 -74 -7):

Gene mutation (Bacterial reverse mutation assay / Ames test): S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. Coli WP2 uvrA were all negative with and without metabolic activation (OECD 471, RL1)

Cytogenicity (micronucleus assay): negative (OECD 487, RL2)

Gene mutation (mammalian cells / TK test): negative (OECD 476, RL1)

In vivo:

no data available and no further data needed

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental phases of the study were performed between 07 January 2010 and 15 February 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
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
GLP compliance:
yes (incl. certificate)
Remarks:
The department of health of the government of the United Kingdom
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Type and identity of media: RPMI 1640
Properly maintained: yes
Periodically checked for Mycoplasma contamination: yes
Periodically checked for karyotype stability: no
Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and beta-naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
A maximum dose level of 5000 µg/mL, the maximum recommended dose level, was used.

Vehicle and positive controls were used in parallel with the test material. Solvent (R0 medium) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) Sigma batch 142314732109252 at 400 µg/mL and 150 µg/mL for the 4-hour and 24-hour exposures respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0164185 at 2 µg/mL was used as the positive control in the presence of metabolic activation.
Vehicle / solvent:
Vehicle used: RPMI 1640 without serum (R0)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Vehicle (R0 medium) treatment groups were used as the vehicle controls.
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Vehicle (R0 medium) treatment groups were used as the vehicle controls.
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours with and without S9 mix, and 24 hours without S9 mix
- Expression time (cells in growth medium): 2 days after the end of the treatment, cells were plated for determination of the cloning efficiency and the mutation frequency in 96-well microtitre plates containing TFT selective medium. The microtitre plates were incubated for 10 to 14 days.
- Selection time: 10 - 14 days

SELECTION AGENT: 4 µg/mL 5-trifluorothymidine (TFT)

STAIN: MTT (2.5 mg/mL in PBS)

NUMBER OF REPLICATIONS: duuplicates each in two independent experiments in 96-well microtitre plates

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER: Small and large colonies were differentiated, as small colonies are capable to indicate chromosomal mutations.


Evaluation criteria:
For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value.
IMF has to exceed 126 E6 in order to be considered as positive.
However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds the value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test material induces modest reproducible increases in the mutation frequencies that do not exceed the value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.
Statistics:
The experimental data was analysed using a dedicated computer program which follows the statistical guidelines recommended by the UKEMS.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 3750 µg/mL onward for 4h treatment + S9 and 24h treatment -S9; at 5000 µg/mL for 4 hour treatment - S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no marked changes when test material was dosed into media
- Effects of osmolality: osmolality did not increase by more than 50 mOsm
- Evaporation from medium: no
- Water solubility: slightly soluble
- Precipitation: at and above 312.5 µg/mL
- Definition of acceptable cells for analysis: Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick.

RANGE-FINDING/SCREENING STUDIES: The dose range used in the preliminary toxicity test was 19.53 to 5000 µg/ml for all three of the exposure groups. In the 4-hour exposure group in the absence of metabolic activation there were no marked dose related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle control. However, a modest reduction was observed in the 4-hour exposure group in the presence of metabolic activation, and a much more marked reduction was observed in the 24-hour exposure group. A precipitate of the test material was observed at and above 19.53 µg/ml in all three of the exposure groups. In the subsequent mutagenicity test the maximum dose level for all three of the exposure groups was the maximum recommended dose level of 5000 µg/ml.

HISTORICAL CONTROL DATA
- Positive historical control data: Mutation frequencies ranged from 466.26 to 2223.55
- Negative (solvent/vehicle) historical control data: Mutation frequencies ranged from 65.97 to 184.74

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

Please see Attached "Tables 1 to 10"

Due to the nature and quantity of tables it was not possible to insert them in this section.

Conclusions:
The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Feb - 9 Jul 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted Jul 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon (S. typhimurium strains)
trp operon (E. coli strain)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
First and second experiment: 313, 625, 1250, 2500 and 5000 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: the vehicle is not suspected of chemical reaction with the test substance and is compatible with the survival of the bacteria and the S9 activity
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
with and without metabolic activation
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene, ICR 191 acridine, daunomycin
Remarks:
-S9: sodium azide, 1.5 µg/plate, TA100, TA1535; ICR 191 acridine, 1 µg/plate, TA1537; daunomycine, 6 µg/plate, TA98; 4-nitroquinoline-1-oxide, 2 µg/plate, E. coli; +S9: 2-aminoanthracene, 10 or 50 µg/plate, all strains; B(a)P, 20 µg/plate, TA98, TA100
Details on test system and experimental conditions:
METHOD OF APPLICATION: experiment 1: preincubation; experiment 2: in agar (plate incorporation)

DURATION
- Preincubation period: 20 min (experiment 1)
- Exposure duration: 48 - 72 h (incubation period, experiment 1 and 2)

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn
Evaluation criteria:
A 2 or 2.5 fold increase in the number of revertant colonies per plate over the background (spontaneous revertant frequency) is used as a criterion to distinguish active mutagens from non-mutagenic materials. The presence of dose-response is a further criterion for mutagenic materials.
Statistics:
Mean values and standard deviation were calculated.
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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 examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: slight precipitation was noted at all concentration levels in all strains, however, this did not interfere with the scoring.

RANGE-FINDING/SCREENING STUDIES: A range-finding study was performed to determine the cytotoxicity and solubilty of the test substance. TA 98 and TA 100 were tested at concentrations of 156, 313, 625, 1250, 2500 and 5000 µg/plate, with and without metabolic activation. The highest concentration that showed low cytotoxicity and/or for which precipitation did not interfere with the scoring, was selected as the highest concentration in the main study.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes. Incidentally the mean results fell outside the range of the historical negative and positive control values. In these cases the scores fell within the standard deviation of the mean value.

Table 1: Experiment 1

EXPERIMENT 1 (preincubation test)

S9-mix

Without

Test item (µg/plate)

TA 98

TA 100

TA 1535

TA 1537

E. coli WP2 uvrA

Vehicle control

17 ± 4

83 ± 2

13 ± 6

5 ± 4

17 ± 2

313

18 ± 4

71 ±7

12 ± 5

5 ± 3

14 ± 4

625

16 ± 4

77 ± 8

13 ± 3

4 ± 1

16 ± 2

1250

21 ± 6

86 ± 7

11 ± 3

4 ± 1

15 ± 4

2500

18 ± 3

84 ± 4

7 ± 3

5 ± 3

22 ± 9

5000

18 ± 4

91 ± 3

10 ± 1

6 ± 1

21 ± 6

DNM

925 ± 180

-

-

-

-

SA

-

388 ± 24

383 ± 42

-

-

4NQO

-

-

-

-

1337 ± 236

ICR 191

-

-

-

2096 ± 59

-

S9-mix

With

Test item (µg/plate)

TA 98

TA 100

TA 1535

TA 1537

E. coli WP2 uvrA

Vehicle control

23 ± 7

76 ± 3

7 ± 2

6 ± 2

20 ± 5

313

21 ± 5

84 ± 18

 9 ± 3

8 ± 1

17 ± 2

625

22 ± 3

87 ± 15

13 ± 2

6 ± 1

12 ± 6

1250

15 ± 2

83 ± 21

5 ± 1

5 ± 1

15 ± 4

2500

22 ± 5

84 ± 4

8 ± 3

6 ± 4

16 ± 3

5000

22 ± 4

91 ± 3

7 ± 2

5 ± 1

13 ± 2

2AA

1991 ± 26

1377 ± 24

52 ± 8

138 ± 26

291 ± 16

B(a)P

173 ± 12

667 ± 71

-

-

-

Vehicle control = distilled water

DNM: daunomycine

SA: sodium azide

4NQO: 4-nitroquinoline-N-oxide

ICR191: ICR 191 acridine

2AA: 2-aminoanthracene

B(a)P: benzo(a)pyrene

 

Table 2: Experiment 2

EXPERIMENT 2 (direct incorporation test)

S9-mix

Without

Test item (µg/plate)

TA 98

TA 100

TA 1535

TA 1537

E. coli WP2 uvrA

Vehicle control

10 ± 3

80 ± 14

7 ± 3

13 ± 6

17 ± 2

313

14 ± 4

76 ± 4

7 ± 2

9 ± 4

14 ± 4

625

15 ± 3

76 ± 8

8 ± 2

11 ± 5

16 ± 2

1250

13 ± 2

80 ± 8

8 ± 3

10 ± 5

15 ± 4

2500

17 ± 6

80 ± 14

7 ± 1

8 ± 3

22 ± 9

5000

15 ± 5

82 ± 6

9 ± 2

7 ± 2

21 ± 6

DNM

713 ± 293

-

-

-

-

SA

-

381 ± 15

278 ± 131

-

-

4NQO

-

-

-

-

1337 ± 236

ICR 191

-

-

-

97 ± 17

-

S9-mix

With

Test item (µg/plate)

TA 98

TA 100

TA 1535

TA 1537

E. coli WP2 uvrA

Vehicle control

17 ± 3

88 ± 7

8 ± 3

7 ± 3

14 ± 5

313

18 ± 4

70 ± 10

9 ± 2

5 ± 3

16 ± 5

625

17 ± 1

90 ± 9

9 ± 2

10 ± 2

19 ± 2

1250

20 ± 2

62 ± 20

5 ± 3

6 ± 2

16 ± 3

2500

21 ± 5

71 ± 6

6 ± 2

9 ± 3

21 ± 3

5000

18 ± 2

75 ± 9

7 ± 2

5 ± 2

19 ± 4

2AA

2057 ± 88

1024 ± 197

41 ± 6

96 ± 22

506 ± 26

B(a)P

203 ± 8

509 ± 39

-

-

-

Vehicle control = distilled water

DNM: daunomycine

SA: sodium azide

4NQO: 4-nitroquinoline-N-oxide

ICR191: ICR 191 acridine

2AA: 2-aminoanthracene

B(a)P: benzo(a)pyrene

 

Conclusions:
The test material was considered to be non-mutagenic under the conditions of this test.
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Feb - 27 May 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The duration of the extended treatment was not specified further than lasting 1.5 - 2 cell cycles, no historical vehicle and positive control data.
Justification for type of information:
refer to the analogue justification provided in IUCLID section 13
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
adopted Sep 2014
Deviations:
yes
Remarks:
the duration of the extended treatment was not specified further than lasting 1.5 - 2 cell cycles, historical data is not given
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 supplemented with fetal bovine serum (FBS) to a final concentration of 10% (v/v)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
4 h treatment (with and without metabolic activation): 0.48, 0.8 and 2.4 µg/mL
Extended treatment (no further information on duration, without metabolic activation): 0.48, 0.8 and 2.4 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9: colchicin (no concentration given); +S9: benzo[a]pyrene (no concentration given)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 h and 1.5 - 2 cell cycles, no further information available (the cell cycle duration was determined in negative control cultures, the duration was not reported in the study report)
- Fixation time (start of exposure up to fixation or harvest of cells): 3 h treatment and extended treatment: after 1.5 - 2 cell cycles, no further information available (the cell cycle duration was determined in negative control cultures, the duration was not reported in the study report

STAIN (for cytogenetic assays): Hoechst 33258 solution and Pyronin Y

NUMBER OF REPLICATIONS:2

NUMBER OF CELLS EVALUATED: At least 2000 (1000 cells per culture, two cultures per concentration)

DETERMINATION OF CYTOTOXICITY
- Method: relative increase in cell count (RICC). Relative cytotoxicity = 100% - RICC (%).

OTHER EXAMINATIONS:
- Other: in order to estimate the required cell cycle number and treatment duration (1.5 - 2 cell cycles after treatment start), additional vehicle control cultures were maintained. Two of these cultures were harvested immediately after removing the test substance from the treated cultures and the cells were counted. Approximately 24 h later two untreated cell cultures were harvested for cell counting. Mitosis was shown by cell counting at the end of analysis and comparing this value with the number of cells at the beginning of the treatment.
Evaluation criteria:
A test substance was considered to be genotoxic if at least a 3-fold increase in number of micronuclei in relation to the negative control was counted and/or a dose-response relationship was observed.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: slight precipitation was observed at and above 2.4 µg/mL.

RANGE-FINDING/SCREENING STUDIES: A range-finding study was performed, using the concentrations 0,48, 2.4, 12, 60, 300 and 1500 µg/mL. Due to precipitation in the culture medium, 2.4 µg/mL was selected as the highest concentration in the main study (see Table 1 under 'Any other information on results incl. tables').

COMPARISON WITH HISTORICAL CONTROL DATA: No.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The cytotoxicity was estimated to be 13.2% at the highest concentration of 2.4 µg/mL.

Table 1: Results of main experiment

Test item

Concentration [µg/mL]

Threshold value*

Number of cells with MN (average of 2 cultures)

Exposure period 3h, fixation time after 1.5 – cell cycles**, without S9 mix

Vehicle control

-

13.5

4.5

CH

-

13.5

26.0

Test substance

0.27

13.5

6.0

0.80

13.5

7.5

2.4

13.5

9.0

Exposure period 3h, fixation time after 1.5 – cell cycles**, with S9 mix

Vehicle control

-

18.0

6.0

B[a]P

-

18.0

27.0

Test substance

0.27

18.0

9.5

0.80

18.0

10.0

2.4

18.0

9.5

Exposure period 24h, fixation time after 1.5 – cell cycles**, without S9 mix

Vehicle control

-

16.5

5.5

CH

-

16.5

41.0

Test substance

0.27

16.5

4.5

0.80

16.5

7.0

2.4

16.5

9.0

CH: colchicine; B[a]P: benzo[a]pyrene (positive controls)

*the 3-fold increase in micronuclei in comparison with the average

micronuclei in the negative control

**the cell cycle duration was determined in negative control cultures,

the duration was not reported in the study report

Conclusions:
The test material did not induce any toxicologically significant increases in the number of cells with micronuclei and is therefore considered to be non-clastogenic and non-aneugenic under the conditions of the test.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Reason / purpose:
read-across source
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 3750 µg/mL onward for 4h treatment + S9 and 24h treatment -S9; at 5000 µg/mL for 4 hour treatment - S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
Since no toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells are observed, the test material is considered to be non-mutagenic under the conditions of the test.
Executive summary:

The source substance (pentacalcium hydroxide tris(orthophosphate) is considered to be non-mutagnic as shown in the HPRT test. As explained in the justification for type of information, the differences in molecular structure between the target and the source are unlikely to lead to differences in genetic toxicity.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Reason / purpose:
read-across source
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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 examined
Positive controls validity:
valid

   

Conclusions:
The constituent indicated in the test material information is considered to be non-mutagenic under the conditions of this test.
Executive summary:

The source substance (pentacalcium hydroxide tris(orthophosphate) is considered to be non-mutagnic as shown in the Ames test. As explained in the justification for type of information, the differences in molecular structure between the target and the source are unlikely to lead to differences in genetic toxicity.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Reason / purpose:
read-across source
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
Since no toxicologically significant increases in the number of cells with micronuclei were found, it is considered that the test material is non-clastogenic and non-aneugenic under the conditions of the test.
Executive summary:

The source substance (pentacalcium hydroxide tris(orthophosphate) is considered to be non-mutagnic as shown in the in vitro micronucleus test. As explained in the justification for type of information, the differences in molecular structure between the target and the source are unlikely to lead to differences in genetic toxicity.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Since all in vitro genetic toxicity test were negative no further in vivo testing is required according to Annex VIII (8.4) of the REACh regulation.

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

One gene mutation test in bacteria and yeast is available (Brusick, 1976) with tricalcium bis(orthophosphate) which is not sufficient for assessment because the study is missing S. typhimurium strain TA 102 or a DNA-proficient strain of E.coli in order to detect cross-linking mutagens and only 3 strains of S. typhimurium were tested (TA 1535, TA 1537, TA 1538). Nevertheless it supports the findings found with the structural analogue pentacalcium tris(ortho)phosphate. This test was performed with Saccharomyces cerevisae strain D4 and three S. typhimiurium strains (TA 1535, TA 1537 and TA 1538) with and without metabolic activation. Metabolic activation systems were derived from 3 different organs and species: liver, lung and testes of mice, rats and primates, respectively. 0.00053%, 0.000265% and 0.0001325% of the test substance were used to evaluate the mutagenic potential. In all systems and at all concenration no mutagenic potential was observed.

No further data is available with tricalcium bis(orthophosphate).

Genetic toxicity with the read across substance pentacalcium tris(ortho)phosphate was evaluated in three different in vitro tests. Due to structural similarity of calcium phosphates as described in the analgoue justification a similar outcome for tricalcium bis(orthophosphate) is assumed.

Genetic toxicity (mutagenicity) in bacteria in vitro with pentacalcium tris(orthophosphate):

The gene mutation test in bacteria was performed according to OECD 471. The test substance was tested for mutagenic activity in Salmonella typhimurium strains TA98, TA100, TA 1535 and TA1537 and E. coli WP2 uvrA at concentrations ranging from 313 to 5000 µg/plate. The tests were conducted, using the preincubation method, on agar plates in the presence and absence of an Aroclor 1254 induced rat liver preparation and co-factors (S9 mix). Positive control compounds demonstrated the sensisitivty of the assay and the metabolising potential of the S9 mix. No significant increase in the number of revertants was observed in any of the 5 bacterial strains, in either activation condition, in the main and the confirmatory test. Slight precipitation was observed in all test material concentrations which did not influence the results of the assay. No cytotoxicity was observed up to highest concentration tested. Therefore, the test material was considered to be non-mutagenic under the conditions of the test.

Genetic toxicity (cytogenicity) in mammalian cells in vitro pentacalcium tris(orthophosphate):

An in vitro Micronucleus Test according to OECD TG 487 and in compliance with GLP was performed with pentacalcium hydroxide tris(orthophosphate). Chinese hamster lung fibroblasts (V79) were treated in duplicate cultures with the test material or vehicle (RPMI 1640 medium supplemented with 10% (v/v) FBS) in the absence or presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix ). Short-term (4 hours with and without S9 mix) and long-term (1.5 to 2 normal cell cycles without S9 mix; duration not given in the report) experiments were conducted at concentrations of 0.48, 0.80 and 2.4 µg/mL. Fixation of the cells was performed 24 hours (approx. 1.5 to 2.0 normal cell cycles) after start of exposure with the test material. Appropriate solvent and positive controls were included in the test and gave the expected results. The number of micronucleated cells found after treatment with the test item was within the normal range of the negative control and thus no genotoxic effects were recorded either in the presence or in the absence of metabolic activation. Slight precipitation was observed at 2.4 µg/mL. The cytotoxicity was estimated to be 13.2% at the highest concentration of 2.4 µg/mL. Based on the results of the study the test material is considered not to be clastogenic or aneugenic under the conditions of this in vitro study.

Genetic toxicity (mutagenicity) in mammalian cells in vitro pentacalcium tris(orthophosphate):

An in vitro Mammalian Cell Gene Mutation Test was performed with pentacalcium hydroxide tris(orthophosphate) in mouse lymphoma L5178Y cells (heterozygous at the thymidine kinase locus) according to OECD TG 476 and under GLP. The cells were treated with the test substance in duplicate cultures, together with vehicle (RPMI 1640 medium without serum) and positive controls (single cultures). The cells were exposed to the test substance for 4 hours in the absence and presence of metabolic activation (phenobarbital and beta-naphthoflavone-induced rat liver S9-mix) in the main and the confirmatory test. The concentration range of the test material in both experiments was 312.5 to 5000 µg/mL following the results of a preliminary toxicity test. Precipitations were seen at and above 312.5 µg/mL. The vehicle controls were within the normal range for the L5178Y cell line at the TK locus. The positive controls induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. Cytotoxicity was observed from 3750 µg/mL onward for 4h treatment + S9 mix and 24h treatment without S9 mix and at 5000 µg/mL for 4 hour treatment without S9 mix. Thus, since the test material did not induce any toxicological significant increases in mutant frequencies up to the highest concentration of 5000 µg/mL it was considered to be non-mutagenic to L5178Y cells under the conditions of the test.


Endpoint Conclusion: All in vitro tests showed negative results with the read across substance pentacalcium tris(orhtophosphate) which indicate no genotoxic potential for that substance and tricalcium bis(orthophosphate). A negative gene mutation test in bacteria and yeast with tricalcium bis(orthophosphate) supports this conclusion.

Since all in vitro test showed clear negative results it is considered scientifically unjustified to do any further testing to assess the genotoxicity and mutagenicity of tricalcium bis(orthophosphate) in vivo. This is in line with Annex VIII (8.4) of the REACh regulation as only if there are positive results in any of the in vitro genotoxicity studies an appropriate in vivo mutagenicity study shall be considered.

Justification for classification or non-classification

The available data on genetic toxicity do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.