Calcium hydrogenorthophosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No reliable data is available with calcium hydrogenorthophosphate. Reliable data is available with the read across substance pentacalcium hydroxide tris(orthophosphate).

In vitro (RA-A 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

Reference

Reference 1

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 for cross-reference:

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.

Reference 2

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 for cross-reference:

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.

Reference 3

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 for cross-reference:

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 is available (Fujita, 1987) with calcium hydrogenorthophosphate which is not sufficient for assessment because only the abstract is available in English and only two strains were tested. The test substance was tested for mutagenic activity in Salmonella typhimurium strains TA97 and TA102 at concentrations ranging from 100 to 10000 µg/plate. The tests were conducted with and without metabolic activation. No significant increase in the number of revertants was observed in any of the two bacterial strains, in either activation condition. Under these test conditions calcium hydrogenorthophosphate is not mutagenic.

One chromosome aberration study is available (Ishidate, 1988) with calcium hydrogenorthophosphate which is not sufficient for assessment because the study is missing testing with metabolic activation and a positive control. Apart from that the study was performed similar to OECD 473 with chinese hamster lung fibroblasts. The cells were treated with 0.125, 0.25, and 0.5 mg/mL calcium hydrogenorthophosphate for 24 and 48 hours. After 24 and 48 hours no structural chromosome aberrations and no increase in polyploidy were induced by calcium hydrogenorthophosphate. Under these test conditions calcium hydrogenorthophosphate is neither clastogenic nor aneugenic.

No further data is available with calcium hydrogenorthophosphate.

Genetic toxicity with the read across substance pentacalcium tris(ortho)phosphate was evaluated in three different in vitro tests.

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 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 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 calcium hydrogenorthophosphate. A negative gene mutation test in bacteria and a chromosome aberration test with calcium hydrogenorthophosphate, although not sufficient for assessment, indicate a non-genotoxic property of calcium hydrogenorthophosphate.

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 calcium bis(dihydrogenorthophosphate) 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.