Pentacalcium hydroxide tris(orthophosphate)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 July 2014 - 15 August 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Pentacalcium hydroxide tris(orthophosphate) is an inorganic salt of the alkaline earth metal calcium and orthophosphate. The water solubility of pentacalcium hydroxide tris(orthophosphate) is low (6.57 mg/L). Dermal absorption is therefore anticipated to be low (ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7c: Endpoint specific guidance. Version 2.0, November 2014). Based on the identity/chemical structure and physicochemical properties, testing for skin sensitisation by means of a Local Lymph Node Assay (OECD 429) is considered to be inappropriate, as it may underestimate the skin sensitising potential of the test substance, leading to a false negative result, due to a low dermal absorption and hence low exposure. For this reason, the Guinea Pig Maximization Test, which involves intradermal injection of the test substance for induction thus ensuring exposure beneath the skin surface, is considered to be the most appropriate method for assessing the skin sensitising potential of this particular substance.

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:

Calcium hydroxycalcium(1+) phosphate (4:1:3)

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles, River, Germany
- Age at study initiation: 4-6 weeks
- Weight at study initiation: 300.9-327.1 g
- Housing: test animals were housed in groups of up to ten
- Diet: commercial feeding mixture (mühle Knull, Rostock, Germany), ad libitum
- Water: tap water (drinking quality, supplemented with 1g/L vitamin C)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3 °C
- Humidity (%): 30-70
- Air changes (per hr): 16
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

water

Concentration / amount:

0.5%

Day(s)/duration:

7

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

100%

Day(s)/duration:

2

Adequacy of induction:

non-irritant substance, but skin pre-treated with 10% SDS

Challenge

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

100%

Day(s)/duration:

1

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

5 (negative control)
10 (test groups)

Details on study design:

RANGE FINDING TESTS:
The appropriate test material concentrations for intradermal and epicutaneous induction and epicutaneous challenge exposures were determined in preliminary test using 6 additional FCA-treated animals.
For intradermal exposure, animals were given the test material at 5, 2.5, 1 and 0.5% suspensions in water by intradermal injections (0.1 mL). Animals were examined for signs of skin irritation at 24 and 48 h post-injection according to the Magnusson Kligman grading scale.
For topical exposure, animals were treated with the test material at 100, 50 and 25% in distilled water for 24 h under occlusive conditions. Irritation responses were assessed at 24 and 48 h after patch substance removal.
Based on the results of preliminary test (see Table 1), in the main test, 0.5% test material in water and 100% test material moistened with water were selected for intradermal and topical treatment, respectively.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous, respectively)
- Exposure period: single injection (intradermal) and 48 h (epicutaneous)
- Test groups:
Intradermal (3 pairs of injections, 0.1 mL):
Injection 1: 1:1 mixture (v/v) FCA/water
Injection 2: 0.5% test substance in water
Injection 3: 0.5% test substance in a 1:1 mixture (v/v) FCA/water
- Negative control group:
Intradermal (3 pairs of injections, 0.1 mL)
Injection 1: 1:1 mixture (v/v) FCA/water
Injection 2: water
Injection 3: 1:1 mixture (v/v) FCA/water

Epicutaneous:
- Test group: test substance at 100%
- Negative control: water

- Site: scapular region (intradermal + epicutaneous)
- Frequency of applications: single
- Duration: Days 0-8 (on day 6, one day prior to epicutaneous induction, the shorn skin of all animals in each group was treated with 0.5 mL of 10% sodium lauryl sulphate vaseline, in order to create a local irritation).

B. CHALLENGE EXPOSURE
- No. of exposures: 1 (challenge)
- Day(s) of challenge: 21 (challenge)
- Exposure period: 24 h
- Test groups: 100% test substance moistened with water
- Control group: 100% test substance moistened with water
- Site: flank region
- Concentrations: 100%
- Evaluation (hr after challenge): 48 and 72 h

OTHER:

Positive control substance(s):

yes

Remarks:

hexyl cinnamic acid (CAS No 101-86-0, routinely evaluated every 6 month at challenge concentrations of 55% in vasseline)

Results and discussion

Positive control results:

Hexyl cinnamic acid (at challenge concentration of 55% in vaseline) induced skin sensitisation reactions in 90% of the treated animals.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

induction (intradermal): 0%; induction (epicutaneoues): 0%, challenge 100%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

no visible clinical symptoms over the period of observation

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

induction (intradermal): 0%; induction (epicutaneoues): 0%, challenge 100%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

no visible clinical symptoms over the period of observation

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no visible clinical symptoms over the period of observation

Remarks on result:

no indication of skin sensitisation

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no visible clinical symptoms over the period of observation

Remarks on result:

no indication of skin sensitisation

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

55%

No. with + reactions:

9

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

55%

No. with + reactions:

9

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

Animal weights

Table 3: Individual animal weights (g) at start / test end (test group)

Animal	Test start	Test end	Body weight change
1	305.3	387.4	82.1
2	313.5	379.8	66.3
3	318.7	414.7	96.0
4	309.3	388.8	79.5
5	301.2	399.9	98.7
6	315.7	385.2	69.5
7	304.1	399.4	95.3
8	321.0	422.8	101.8
9	309.8	375.7	65.9
10	309.3	411.5	102.2

 

Individual weight of control group

Table 4: Individual animal weights (g) at test start and at test end (control group)

Animal	Test start	Test end	Body weight change
K1	309.4	383.0	73.6
K2	300.9	376.1	75.2
K3	324.0	396.1	72.1
K4	327.1	408.5	81.4
K5	326.4	398.7	72.3

 

Table 5: Skin reactions of test animals after treatment with the test material

Animal	Numerical grading after
	24 h	48 h
1	0	0
2	0	0
3	0	0
4	0	0
5	0	0
6	0	0
7	0	0
8	0	0
9	0	0
10	0	0

 

Table 6: Skin reactions of control animals after treatment with the test material

Animal	Numerical grading after
	24h	48h
K1	0	0
K2	0	0
K3	0	0
K4	0	0
K5	0	0

 

Table 7: Skin reactions of animals after challenge treatment with HCA 55 % in vaseline

Animal	Numerical grading after
	24 h	48 h
1	1	1
2	0	0
3	1-2	1-2
4	1	1
5	1-2	2
6	1	1
7	1-2	1-2
8	0-1	1
9	1-2	1
10	1	1

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test material did not induce any skin reactions in intradermally and topically induced guinea pigs after challenge treatment. Therefore, the material does not fulfil the criteria for classification according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), and is thus considered to be not skin sensitizing.