Copper di(acetate)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with Good laboratory Practice and internationally accepted guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

An LLNA study was not conducted because this method is prone to give false positive results for copper compounds and strong dermal irritants.

Test material

Test material information

Constituent 1

Reference substance name:

Copper di(acetate)

EC Number:

205-553-3

EC Name:

Copper di(acetate)

Cas Number:

142-71-2

Molecular formula:

Cu(CO2CH3)2

IUPAC Name:

copper di(acetate)

Details on test material:

- Name of test material (as cited in study report): Copper acetate monohydrate
- Analytical purity: 99.9%
- Lot/batch No.: 11.6.1SD
- Storage condition of test material: room temperature, dark

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 (F-69592 L’ARBRESLE)
- Age at study initiation: 4 weeks old
- Weight at study initiation: between 230 g and 260 g.
- Housing: The animals were housed either in groups of 2 or individually in polycarbonate containers, the flooring of which was covered with dust-free cuttings and the top fitted a stainless steel lid with a feeding device and drinking device of 500 mL.
- Diet (e.g. ad libitum): ad libitum.
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: A minimum acclimatization period of 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C.
- Humidity (%): 30 to 70%.
- Air changes (per hr): Approximately fifteen changes per hour.
- Photoperiod (hrs dark / hrs light): Twelve hours continuous light (07.00 to 19.00) and twelve hours darkness.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

other: Olive oil for the intradermal injections and in liquid paraffin for the topical applications.

Induction 2

Concentration / amount:

8.75% and 4%

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: Olive oil for the intradermal injections and in liquid paraffin for the topical applications.

Challenge 2

Concentration / amount:

8.75% and 4%

No. of animals per dose:

GROUP 1 (negative control) : 5 female guinea pigs.
GROUP 2 (treated) : 10 female guinea pigs.

Details on study design:

RANGE FINDING TESTS:

Determination by intradermal injection of the Maximal Non Necrotizing Concentration (MNNC):

This test was conducted for the purpose of defining a MNNC of the test item which, on intradermal injection during the induction phase, does not risk causing too great a lesion (non-necrotizing concentration), should be well-tolerated systemically and should be the highest to cause mild-to-moderate skin irritation. Two animals received on both sides of the spine, a volume of 0.1 mL of the test item, at four concentrations: diluted at 5%, 2%, 1% and 0.5% in olive oil in order to determine the MNNC. A macroscopic evaluation of the cutaneous reactions was conducted 24 hours after injection.
Due to the fact that necrosis was noted at the four concentrations, the same animals received on both sides of the spine, a volume of 0.1 mL of the test item, at 3 concentrations: diluted at 0.1%, 0.05% and 0.01% in olive oil in order to determine the MNNC. A macroscopic evaluation of the cutaneous reactions was conducted 24 hours after injection.

Determination by topical application ofthe Pre-Maximal Non Irritant Concentration (Pre-MNIC):

This test, which allowed to evaluate the irritantpotential of the test item, defined whether an application of sodium lauryl sulfate would be needed during topical induction phase. The test item was applied on the dorso-lumbar zone of one guinea pig shorn beforehand, with occlusive dressing for 24 hours, at 4 different concentrations: diluted at 70%, 35%, 17.5% and 8.75% in liquid paraffin. A macroscopic evaluation of the cutaneous reactions was conducted 24 hours after removal of the dressing.


Determination by topical application of the Maximal Non Irritant Concentration (MNIC):

This test was carried out for the purpose of determining the MNIC of the test item without risk of an irritant effect during the challenge phase. Three guinea pigs were treated according to the same treatment as animals from GROUP 1 (negative control) for the induction phase (i.e. olive oil and liquid paraffin). During the challenge phase, the animals were treated with the test item placed onto the selected treatment sites and covered with an occlusive dressing for a period of 24 hours at 4 different concentrations: diluted at 70%, 35%, 17.5% and 8.75% in liquid paraffin. A macroscopic evaluation of the cutaneous reactions was conducted 24 and 48 hours after removal of the occlusive dressing and rinse with liquid paraffin.

MAIN STUDY:

A. INDUCTION EXPOSURE

Intradermal Induction:

Day 0
After shearing the scapular zone, three (3) pairs of intradermal injections (ID) of 0.1 ml were performed on the scapular zone in such a way as an injection on each pair is placed to either side of the spine as follows:

GROUP 1 (Negative control):
- 2 ID: Freund’s Complete Adjuvant diluted at 50 % in isotonic sodium chloride;
- 2 ID: olive oil;
- 2 ID: a mixture with equal volumes v/v: Freund’s Complete Adjuvant at 50% and olive oil.

GROUP 2 (Treated):
- 2 ID: Freund’s Complete Adjuvant diluted by 50 % in isotonic sodium chloride;
- 2 ID: test item at 0.1% in olive oil;
- 2 ID: a test mixture in equal volumes v/v: Freund’s Complete Adjuvant at 50% and the test item at 0.2% in olive oil.

Topical Induction:

Day 6
The scapular zone of all the animals in each group, shorn beforehand, was brushed with a solution of sodium lauryl sulfate at 10% in thick vaseline, in order to create a local irritation.

Day 7
A topical application under occlusive dressing for 48 hours was performed on the injection sites of each animal.
- GROUP 1 (Negative control): 0.5 mL of liquid paraffin.
- GROUP 2 (treated): 0.5 mL of the test item at 70%.

Day 9
Occlusive dressing removal and rinse with distilled water and liquid paraffin.

B. CHALLENGE EXPOSURE

Day 20
The experimental procedure of thisphase was identical for both groups GROUP 1 (Negative control) and GROUP 2 (Treated) submitted to this experimentation: on the previously shorn dorso-lumbar zone, an application on either side of the spine, under occlusive dressing, was performed for 24 hours:
- 1 sample cup containing the test item diluted at 8.75% (MNIC);
- 1 sample cup containing the test item diluted at 4%.

Day 21
Occlusive dressing removal and rinse with liquid paraffin.

Positive control substance(s):

yes

Remarks:

α-Hexylcinnamaldehyde

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

8.75%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 8.75%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None.

Results 2

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

4%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 4%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None.

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

8.75%

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

A slight erythema was seen in 10% (1/10) of the animals from the treated group.

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 8.75%. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: A slight erythema was seen in 10% (1/10) of the animals from the treated group..

Results 4

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

4%

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

A slight erythema was seen in 10% (1/10) of the animals from the treated group.

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 4%. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: A slight erythema was seen in 10% (1/10) of the animals from the treated group..

Results 5

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

8.75%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 8.75%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None.

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

4%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 4%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None.

Results 7

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

8.75%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 8.75%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.

Results 8

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

4%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None.

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 4%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None..

Any other information on results incl. tables

PRELIMINARY STUDIES:

MNNC determination:

Necrosis has been observed with the concentrations of 5%, 2%, 1% and 0.5%. No necrosis has been observed with the concentration of 0.1%. In addition, no evidence of irritation was noted at a concentration of 0.1% but as this was the next concentration in the concentration series, this was selected for use in the intradermal induction. The first induction of the Group 2 has been carried out by intradermal injection at the same concentration of 0.1% as maximum non necrosing concentration.

Pre MNIC determination:

24 hours after the removal of the occlusive dressings, no skin reaction was recorded on the treated area whatever the tested concentrations. In view of these results, the concentration selected was 70% for the 2nd induction of the Group 2 and the MNIC determination began at the concentration of 70%.

MNIC determination:

24 and 48 hours after the removal of the occlusive dressings, well defined erythema was recorded on the treated area at 70% in the three animals, well defined erythema was recorded on the treated area at 35% and 17.5% in one animal. In view of this result, the concentrations selected were 8.75% (MNIC) and 4% for the challenge phase.

MAIN TEST:

Induction phase Group 1:

The induction phase was performed by intradermal injection on Day 0 with olive oil and by topical application on Day 7 with liquid paraffin. No cutaneous reaction was recorded after the first induction phase. During the second induction, dryness was noted in 5 animals (5/5), 24 hours after the removal of the occlusive dressing. See Table 1 (attached).

Induction phase Group 2:

The induction phase was performed by intradermal injection on Day 0 with the test item at 0.1% in olive oil and by topical application on Day 7 with the test item at 70% in liquid paraffin. No cutaneous reaction was recorded after the 1^stinduction phase. During the second induction, scab formation was noted in 7 animals (7/10) and slight erythema was noted in 3 animals (3/10), 24 hours after the removal of the occlusive dressing. See Table 1 (attached).

Challenge phase Groups 1 & 2:

The test item has been used diluted at 8.75% and 4% in liquid paraffin.

SENSITISING POTENTIAL ASSESSMENT:

Overall results of the challenge phase with the test item (readings at 24 and 48 hours) are given in Table 2 (attached). Individual scores of macroscopic evaluations performed during challenge phase with the test item are given in Table 3 (attached). The Sensitizing response indices (severity and incidence) are presented in Table 4.

A slight erythema was seen in 10% (1/10) of the animals from the treated group, 24 hours after the challenge phase, on the treated area with the test item at 8.75%. No cutaneous reaction was recorded at 48 hours after the challenge phase. No cutaneous intolerance reaction was recorded in animals from the negative control group after the challenge phase, on the treated area with the test item at 8.75%.

A slight erythema was seen in 10% (1/10) of the animals from the treated group, 24 hours after the challenge phase, on the treated area with the test item at 4%. No cutaneous reaction was recorded at 48 hours after the challenge phase. No cutaneous intolerance reaction was recorded in animals from the negative control group after the challenge phase, on the treated area with the test item at 4%.

WEIGHT EVOLUTION:

The weight growth of negative control animals (Group 1) and treated animals (Group 2) is respectively presented in Tables 5 and 6 and in Figure 1. No abnormality was recorded in the body weight gain of both groups.

MORTALITY:

No mortality was registered during the main test.

CONCLUSION:

It was concluded that the test item is not a skin sensitizer.

 

 

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Copper acetate monohydrate is not a skin sensitiser.
Classification according to Directive 67/548/EEC: Not classified.
Classification according to CLP/GHS: Not classified.

Executive summary:

A GLP-compliant Guinea Pig Maximisation Test was carried out in accordance with OECD Guideline 406 and EU Method B.6. After induction (intradermal injection at 0.1% and topical application at 70%) of 10 Guinea Pigs with the test item Copper acetate monohydrate and a 10-day rest phase, the challenge phase, under occlusive dressing for 24 hours, consisted of a single topical application of the test item diluted at 8.75% and at 4% in liquid paraffin.

It was only recorded a slight erythema in 10% (1/10) of the animals from the treated group, 24 hours after the challenge phase, on the treated area with the test item at 8.75%. No cutaneous reaction was recorded at 48 hours after the challenge phase. No cutaneous intolerance reaction was recorded in animals from the negative control group after the challenge phase, on the treated area with the test item at 8.75%.

A slight erythema was seen in 10% (1/10) of the animals from the treated group, 24 hours after the challenge phase, on the treated area with the test item at 4%. No cutaneous reaction was recorded at 48 hours after the challenge phase. No cutaneous intolerance reaction was recorded in animals from the negative control group after the challenge phase, on the treated area with the test item at 4%.

It was concluded that copper acetate monohydrate is not sensitising to skin.