reaction mass of calcium bis(dihydrogenorthophosphate) and calcium hydrogenorthophosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No reliable data is available with the reaction mass of calcium bis(dihydrogenorthophosphate) and calcium hydrogenorthophosphate. Reliable data is available with the read across substance pentacalcium hydroxide tris(orthophosphate).

In vitro 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 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:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category

(1) All salts are inorganic phosphates, composed of a phosphate anion and a calcium cation.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Ca2+ cation and the PO43-anion.
(3) Orthophosphate salts of these types are not considered to differ in their systemic toxicity profile; differences arise in their local effects profile due to the increasing or decreasing acidity of the substances. This has been shown not to have an effect on the systemic toxicity profile of the substances, thus suggesting that they are metabolized via similar metabolic pathways and to similar breakdown products. A number of studies are provided to show that calcium inorganic orthophosphates exhibit low systemic toxicity via the oral route for acute exposure. . These data are provided in Section 7.2 and Section 7.5 of this dossier. The information provided in these records is considered to be indicative of a group of chemicals that are likely to behave in a similar way in vivo.


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

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.

Reference 2

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:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category

(1) All salts are inorganic phosphates, composed of a phosphate anion and a calcium cation.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Ca2+ cation and the PO43-anion.
(3) Orthophosphate salts of these types are not considered to differ in their systemic toxicity profile; differences arise in their local effects profile due to the increasing or decreasing acidity of the substances. This has been shown not to have an effect on the systemic toxicity profile of the substances, thus suggesting that they are metabolized via similar metabolic pathways and to similar breakdown products. A number of studies are provided to show that calcium inorganic orthophosphates exhibit low systemic toxicity via the oral route for acute exposure. . These data are provided in Section 7.2 and Section 7.5 of this dossier. The information provided in these records is considered to be indicative of a group of chemicals that are likely to behave in a similar way in vivo.


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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:
Vehicle (R0 medium) treatment groups were used as the vehicle controls.


Justification for choice of vehicle:
Formed a suspension suitable for dosing

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:

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476) and Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

One main experiment was performed. In this main experiment, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at six dose levels, in duplicate, together with vehicle (R0 medium) and positive controls. The exposure groups used were as follows: 4 hour exposures both with and without metabolic activation, and 24 hours without metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test and was 312.5 to 5000 µg/ml for all three of the exposure groups.

The maximum dose level used was the maximum recommended dose level (5000 µg/ml). Precipitate of the test material was observed at and above 312.5 µg/ml in all three of the exposure groups. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in any of the three exposure groups.

Evaluation criteria:

Please see Interpretation of Results in "Any other information and methods incl. tables" section. As this section will not accommodate the required information.

Statistics:

Please see "Any other information and methods incl. tables" section.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

non-mutagenic

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS

Preliminary Toxicity Test

The dose range of the test material used in the preliminary toxicity test was 19.53 to 5000 µg/ml.

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.

Mutagenicity Test

A summary of the results from the test is presented in attached Table 1.

4-Hour Exposure With and Without Metabolic Activation

The results of the microtitre plate counts and their analysis are presented in attached Tables 2 to 7.

As was seen in the preliminary toxicity test, there was evidence of dose related toxicity following exposure to the test material in the presence of metabolic activation, as indicated by the %RSG and RTG values. However, the toxicity observed in the presence of metabolic activation was more marked than that observed in the preliminary toxicity test and near optimum levels of toxicity were achieved (Table 6). On this occasion there was also evidence of modest levels of toxicity in the absence of metabolic activation (Table 3). There was no evidence of any reductions in viability (%V), therefore indicating that no residual toxicity had occurred in either the absence or presence of metabolic activation. Acceptable levels of toxicity were seen with both positive control substances (Tables 3 and 6).

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 3 and 6).

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 3 and 6).

The numbers of small and large colonies and their analysis are presented in Tables 4 and 7.

24-Hour Exposure Without Metabolic Activation

The results of the microtitre plate counts and their analysis are presented in attached Tables 8 to 10.

As was seen in the preliminary toxicity test, once again there was evidence of marked dose related toxicity following exposure to the test material, as indicated by the %RSG and RTG values, and near optimum levels of toxicity were achieved (Table 9). There was no evidence of any reductions in viability (%V), therefore indicating that no residual toxicity had occurred. The positive control induced acceptable levels of toxicity (Table 9).

The 24-hour exposure without metabolic activation demonstrated that the extended time point had a marked effect on the toxicity of the test material.
The vehicle control mutant frequency value was within the acceptable range of 50 to 200 x 10-6 viable cells. The positive control produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily (Table 9).

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell (Table 9).

The numbers of small and large colonies and their analysis are presented in Table 10.

Remarks on result:

other: strain/cell type: Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line

Remarks:

Migrated from field 'Test system'.

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:

Interpretation of results:
negative Non-mutagenic

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.
This study has been selected as the key study because the results are sufficient in order to derive a reliable conclusion on classification and labelling in accordance with Regulation EC (No.) 1272/2008 (EU CLP).

JUSTIFICATION FOR READ ACROSS;

The following substances are considered to be similar enough to facilitate read across for genetic toxicity endpoints:
- calcium bis(dihydrogenorthophosphate): EC No. 231-837-1
- calcium hydrogenorthophosphate: EC No. 231-826-1
- tricalcium bis(orthophosphate): EC No. 231-840-8
- pentacalcium hydroxide tris(orthophosphate): EC No. 235-330-6

All of the above substances have exhibited similar toxicity in acute oral and dermal studies.
As the reaction mass of calcium bis(dihydrogenorthophosphate) and calcium hydrogenorthophosphate is a mixture of two calcium orthophosphates it is appropriate to include this substance in the above group. In addition the reaction mass of calcium bis(dihydrogenorthophosphate) and calcium hydrogenorthophosphate has been shown to exhibit the same systemic toxicity profile as the other calcium and magnesium orthophosphates detailed above and on this basis read across is justified.

Read across is justified on the following basis:

1. Low systemic toxicity in in vivo studies.
A number of studies are provided to show that calcium inorganic orthophosphates exhibit low systemic toxicity via the oral route for acute exposure. . These data are provided in Section 7.2 and Section 7.5 of this dossier. The information provided in these records is considered to be indicative of a group of chemicals that are likely to behave in a similar way in vivo.
2. Substance similarities:
All salts are inorganic phosphates, composed of a phosphate anion and a calcium cation. Orthophosphate salts of these types are not considered to differ in their systemic toxicity profile; differences arise in their local effects profile due to the increasing or decreasing acidity of the substances. This has been shown not to have an effect on the systemic toxicity profile of the substances, thus suggesting that they are metabolized via similar metabolic pathways and to similar breakdown products.

Justification for no further testing for genetic toxicity:

As a number of studies exist to show that calcium orthophosphates are not genotoxic it is not considered to be appropriate to conduct a set of in vitro genotoxicity studies on all of the aforementioned substances. In addition the Ca2+ and PO43- ions are essential for life and are not considered to be genotoxic or mutagenic in standard test systems and therefore read across is deemed to be scientifically justified and not further studies are required.

Executive summary:

Introduction. 

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476) and Method B17 of Commission Regulation (EC) No. 440/2008 of30 May 2008.

Methods. 

One main experiment was performed. In this main experiment, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at six dose levels, in duplicate, together with vehicle (R0 medium) and positive controls. The exposure groups used were as follows: 4‑hour exposures both with and without metabolic activation, and 24 hours without metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test and was 312.5 to 5000 µg/ml for all three of the exposure groups.

Results. 

The maximum dose level used was the maximum recommended dose level (5000 µg/ml). Precipitate of the test material was observed at and above 312.5 µg/ml in all three of the exposure groups. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in any of the three exposure groups.

Conclusion. 

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Reference 3

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

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category

(1) All salts are inorganic phosphates, composed of a phosphate anion and a calcium cation.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Ca2+ cation and the PO43-anion.
(3) Orthophosphate salts of these types are not considered to differ in their systemic toxicity profile; differences arise in their local effects profile due to the increasing or decreasing acidity of the substances. This has been shown not to have an effect on the systemic toxicity profile of the substances, thus suggesting that they are metabolized via similar metabolic pathways and to similar breakdown products. A number of studies are provided to show that calcium inorganic orthophosphates exhibit low systemic toxicity via the oral route for acute exposure. . These data are provided in Section 7.2 and Section 7.5 of this dossier. The information provided in these records is considered to be indicative of a group of chemicals that are likely to behave in a similar way in vivo.


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

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 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

No data is available on the substance itself.

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 the reaction mass of calcium bis(dihydrogenorthophosphate) and 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 the reaction mass of calcium bis(dihydrogenorthophosphate) and calcium hydrogenorthophosphate

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 therefore are conclusive but not sufficient for classification. The reaction mass of calcium bis(dihydrogenorthophosphate) and calcium hydrogenorthophosphate is not considered to be genotoxic.