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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key 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) A number of studies are provided to show that monovalent calcium and/or magnesium inorganic orthophosphates exhibit low acute oral toxicity. These data are provided in Section 7.2.1 of this dossier. The information provided in these records is considered to be of suitable relevance and reliability to underpin the read across for the acute dermal and inhalation endpoints. All substances are ionic and will readily dissociate to their ionic forms in aqueous environments. The orthophosphate ion can undergo ionisation with loss of H+ from each of the three –OH groups and therefore can occur in the -1, -2 or -3. The degree of ionisation is dependent upon the associated cation and the ambient pH (if in solution). Calcium and phosphate are key elements in various cellular processes their import and export over cell membranes is regulated via pore systems and usually tightly regulated. Orthophosphate salts of these types are not considered to differ in their systemic toxicity profiles. Differences arise in their local effects profile due differences in pH and buffering capacities. This does not have an effect on systemic toxicity.

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 source
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
08 July 2014 - 15 August 2014
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) A number of studies are provided to show that monovalent calcium and/or magnesium inorganic orthophosphates exhibit low acute oral toxicity. These data are provided in Section 7.2.1 of this dossier. The information provided in these records is considered to be of suitable relevance and reliability to underpin the read across for the acute dermal and inhalation endpoints. All substances are ionic and will readily dissociate to their ionic forms in aqueous environments. The orthophosphate ion can undergo ionisation with loss of H+ from each of the three –OH groups and therefore can occur in the -1, -2 or -3. The degree of ionisation is dependent upon the associated cation and the ambient pH (if in solution). Calcium and phosphate are key elements in various cellular processes their import and export over cell membranes is regulated via pore systems and usually tightly regulated. Orthophosphate salts of these types are not considered to differ in their systemic toxicity profiles. Differences arise in their local effects profile due differences in pH and buffering capacities. This does not have an effect on systemic toxicity.

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

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

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.

Data source

Materials and methods

Test material

Constituent 1
Reference substance name:
Reaction mass of Calcium bis(dihydrogenorthophosphate) and Calcium hydrogenorthophosphate (multi-constituent)
IUPAC Name:
Reaction mass of Calcium bis(dihydrogenorthophosphate) and Calcium hydrogenorthophosphate (multi-constituent)
Test material form:
solid
Details on test material:
Sponsor's identification: A reaction mass of monocalcium phosphate and dicalcium phosphate
Description : cream coloured granular solid
Purity : 82.1% (26.8% dicalcium phosphate and 55.3% monocalcium phosphate)
Batch number : Not supplied
Date received : 05 May 2010
Storage conditions: room temperature in the dark.

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)

Resultsopen allclose all
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
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
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
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
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
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.
Executive summary:

No skin senistisation properties are considered from the source study by Gruemmer, 2014 (GPMT). 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 the partition coefficient that are higher than the typical experimental error of the test method.