Diphosphoric acid, copper salt

Endpoint summary

Description of key information

The test substance is not skin sensitising.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-09-09 to 2014-01-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

In accordance with Annex VII, Section 8.3, Column 2 the use of the Guinea-pig maximisation test (GMPT) of Magnusson and Kligmman (OECD 406) as opposed to the Local Lymph Node Assay (LLNA) is justified on the basis that LLNA studies have been shown to give false positive results for copper and copper compounds and as such the GMPT study is considered to be the most reliable for determining the true sensitisation potential of a copper-based material.

Specific details on test material used for the study:

- Name of test material: Copper(ll)-hydroxide Phosphate
- Physical state: light green solid powder
- Lot/batch No.: 9000027158
- Storage condition of test material: room temperature 15-25 °C
- solid: particulate/powder

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: young adult
- Weight at study initiation: 304-327 g
- Housing: in groups up to 10 animals per cage
- Diet: commercial feeding mixture (Muhle Knull, Rostock, Germany) ad libitum
- Water: tap water (drinking quality, supplemented with 1 g/l vitamin C) ad libitum
- Acclimation period: 5 days

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

Route:

intradermal and epicutaneous

Vehicle:

water

Concentration / amount:

The test material was not soluble in distilled water and was injected as suspension. For topical application the test material was moistened with distilled water (100%) or applied as a suspension (50%, 25%).

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

The test material was not soluble in distilled water and was injected as suspension. For topical application the test material was moistened with distilled water (100%) or applied as a suspension (50%, 25%).

No. of animals per dose:

Test group: 10
Control group: 5
Preliminary test: 6

Details on study design:

RANGE FINDING TESTS:
The appropriate concentrations of the test material and the appropriate vehicle for the intradermal induction phase, topical induction phase and challenge phase were determined on additional 6 Freund complete adjuvant (FCA) treated animals.
Intradermal injection:
The irritation response to intradermal injection of various concentrations of the test substance was examined in three guinea pigs. An area of the flanks was clipped free from hair with electric clippers. Amounts of 0.1 ml of the selected test concentrations (5%; 2.5%; 1%. and 0.5% suspensions of the test material in distilled water) were applied by intradermal injection. 24 and 48 hours after injection the animals were examined for signs of irritation according to the MAGNUSSON AND KLIGMAN GRADING SCALE.
Topical treatment:
The irritation response to topical treatment of various concentrations of the test substance was examined in three further guinea pigs. The flanks of the animals were clipped. Filter paper fully-loaded with the test substance (100%; 50% 25% in distilled water) was attached to the skin of the guinea-pigs and held in contact by an occlusive dressing for 24 hours.
The animals were observed and examined for signs of irritation according to the MAGNUSSON AND KLIGMAN GRADING SCALE approximately 24 hours and 48 hours after removing the test material.

MAIN STUDY
A1. INDUCTION EXPOSURE: intradermal injection (day 0)
- Test groups:
A) 1:1 mixture (v/v) FCA/water
B) Test substance suspension of 0.5% in distilled water
C) Test substance at the selected concentration formulated in a 1:1 mixture (v/v) FCA/water
- Control group: vehicle
- Site: dorsal skin from the scapular region (approximately 4 cm x 6 cm)
- Frequency of applications: once

A2. INDUCTION EXPOSURE: topical application (day 7)
- Exposure period: 48 hours
- Test groups: a patch loaded with 100% finely pulverised test substance moistened with distilled water
- Control group: vehicle
- Site: same as at intradermal injection
- Frequency of applications: once
Because the agent was non-irritating at this concentration, the region was pretreated with 10% sodium lauryl sulphate in vaseline for 24h.

B. CHALLENGE EXPOSURE (day 21)
- Exposure period: 24 hours
- Test groups: a patch loaded with 100% finely pulverised test substance moistened with distilled water
- Control group: vehicle
- Site: one flank of the animals
- Evaluation (hr after challenge): 24 and 48

Positive control substance(s):

no

Positive control results:

The reliability checks are performed regularly with hexyl cinnamic aldehyde (HCA -CAS No. 101-86-0) in the test laboratory (every six months).
The latest reliability check was performed from 04/09/2013 - 11/10/2013.
90% of animals treated with the positive control (HCA - 55% in vaseline) showed a skin reaction with numeral grading from 0-1 up to 2 according to "MAGNUSSON AND KLiGMAN GRADING SCALE FOR THE EVALUATION OF CHALLENGE PATCH TEST REACTIONS". The positive control showed a sensitization effect and the validity of the test system.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100% of the test material moistened with distilled water

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100% of the test material moistened with distilled water

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

vehicle

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

vehicle

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No

Remarks on result:

no indication of skin sensitisation

Interpretation of results:

GHS criteria not met

Conclusions:

The test material Copper(ll)-hydroxide Phosphate is considered to be not skin sensitizing.

Executive summary:

The test material Copper(ll)-hydroxide Phosphate did not induce any skin reactions in intradermally and topically induced guinea pigs after challenge treatment. Therefore, the test 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The skin sensitising potential of copper pyrophosphate was tested in a Guinea Pig Maximization Test (GPMT) carried out in accordance with OECD Guideline 406 and GLP (Grümmer, 2014). Treatment and vehicle control groups consisted of 10 and 5 female Dunkin-Hartley guinea pigs, respectively. Based on the results of preliminary tests on additional animals, the test substance was applied at 1% suspension in water for intradermal induction (causing mild-to-moderate irritation) and at 100% (moistened with water) for topical induction and challenge (maximum non-irritating concentration).

For intradermal induction, animals of the treatment group received three pairs of injections (0.1 mL each) consisting of a 1:1 (v/v) mixture of Freund's complete adjuvant (FCA) and water, test substance suspension and a 1:1 (v/v) mixture of FCA and test substance suspension, respectively. Animals of the control group were treated with vehicle (water). After one week, animals were induced by topical application of the neat test substance (moistened with water) or water for 48 h on skin regions previously treated with 10% sodium lauryl sulphate in Vaseline for 24 h. Two weeks after the last induction application, animals of the treatment group were challenged with the test substance by topical application for 24 h under an occlusive patch. Skin reactions were assessed 24 and 48 after patch removal. Animals of the control group were similarly treated with the vehicle. While this is a deviation from the test method used, based on the outcome below, it is not considered to affect the validity of the test results obtained with the test material.

There were no signs of systemic toxicity or effects on body weight gain during the study period. No skin reactions were observed in any animal at 24 and 48 h after removal of the challenge patch.

The latest reliability check performed at the testing facility less than 6 months prior to this study with hexyl cinnamic aldehyde (5% in paraffin oil for intradermal induction; 75% in Vaseline for topical induction; 55% in Vaseline for challenge) yielded the expected results (90% positive reactions) showing the validity of the test system.

Based on the study results, copper pyrophosphate was considered to be not skin sensitising.

No skin sensitisation was observed in a previous GPMT conducted in male guinea pigs according to OECD Guideline 406 and GLP (Grümmer, 2013). The test substance was prepared as a 0.2 g/mL suspension in water. The test suspension was centrifuged and the saturated supernatant was used for intradermal injections, while for epicutaneous applications the test suspension was used as such. Given the low water solubility of copper pyrophosphate, the test substance concentration used for intradermal induction was likely to be very low to negligible. Therefore, the results of this study are of limited relevance for hazard assessment and thus only used as information supporting the weight of evidence.

The available data indicates that copper pyrophosphate is not a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

Based on available data on skin sensitisation the test item is not classified according to Regulation (EC) No 1272/2008 (CLP).