reaction mass of calcium bis(dihydrogenorthophosphate) and calcium hydrogenorthophosphate

Genetic toxicity: in vitro

Administrative data

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’.

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Pentacalcium hydroxide tris(orthophosphate)

EC Number:

235-330-6

EC Name:

Pentacalcium hydroxide tris(orthophosphate)

Cas Number:

12167-74-7

Molecular formula:

Ca5HO13P3

IUPAC Name:

tricalcium diphosphate

Constituent 2

Reference substance name:

tricalcium phosphate

IUPAC Name:

tricalcium phosphate

Test material form:

solid: particulate/powder

Method

Target gene:

his operon (S. typhimurium strains)
trp operon (E. coli strain)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

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

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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.

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

Conclusions:

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