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Toxicological information

Skin sensitisation

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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: Already evaluated by the Competent Authorities for Biocides and Existing Substance Regulations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
Slight variations of humidity were noted beyond the norms (minimum 45% RH) with aminimum at 36.4% RH. This deviation is not considered to have affected the outcome of the study.
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
yes
Remarks:
Slight variations of humidity were noted beyond the norms (minimum 45% RH) with aminimum at 36.4% RH. This deviation is not considered to have affected the outcome of the study.
Qualifier:
according to
Guideline:
EPA OTS 798.4100 (Skin Sensitisation)
Deviations:
yes
Remarks:
Slight variations of humidity were noted beyond the norms (minimum 45% RH) with aminimum at 36.4% RH. This deviation is not considered to have affected the outcome of the study.
GLP compliance:
yes
Type of study:
Freund's complete adjuvant test
Justification for non-LLNA method:
The available study was conducted prior to the date on whch the LLNA became the method of choice.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test material: Copper II sulphate pentahydrate,
Lot/Batch number: Batch number 844,
Specification: Not stated,
Description: Blue crystals,
Purity: 99.0 - 100.5%,
Stability: Reported to be stable under the conditions of the study.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
Source:Charles River France (Cleon, France)
Age/weight at study initiation: Young adults in weight range 250 - 550 g on the day prior to the first induction application or on the day of the range
finding application.
Number of animals per group: 20 in treated group, 10 in vehicle control. 10 used for preliminary investigations.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
Induction: 0.1% w/v solution of copper II sulphate pentahydrate in water for injection or 10% w/w formulation in water for injection for topical
application.
Challenge: 10% w/w solution of copper II sulphate pentahydrate in water for injection was the maximum non-irritant concentration appropriate for use in the challenge phase.
Challenge
Concentration / amount:
Induction: 0.1% w/v solution of copper II sulphate pentahydrate in water for injection or 10% w/w formulation in water for injection for topical
application.
Challenge: 10% w/w solution of copper II sulphate pentahydrate in water for injection was the maximum non-irritant concentration appropriate for use in the challenge phase.
No. of animals per dose:
10 used for preliminary investigations.
20 in treated group, 10 in vehicle control.
Details on study design:
RANGE FINDING TESTS:
Preliminary investigations were completed to determine a slightly irritant concentration for use in the intradermal induction phase and also for topical application in the induction phase. The preliminary study also examined topical doses for a maximum non-irritant level for use at challenge.

For the preliminary testing the substance was prepared as 28, 14, 5, 1, 0.1 or 0.05% solutions in water for injection or as a 90% (w/w) paste or 45, 10 or 1% (w/w) suspension in water for topical application.

The maximum concentration that could be prepared for intradermal injection was 28% w/w paste formed from powdered test material and water.

MAIN STUDY
A. INDUCTION EXPOSURE
Test material preparation: the test material was prepared as a 0.1% w/w solution in water for injection or in a 50:50 mixture of Freund's complete
adjuvant in water for injection. The third pair of injection sites received the adjuvant preparation alone. For topical administration the test substance was prepared as a 10% (w/w) suspension in water for injection.
No. of exposures: Intradermal injection on day 1 and topical application on Day 8
Frequency of applications:
-Injection
Test groups: 3 pairs of injections. In the anterior pair the adjuvant was injected. In the posterior pair the test material was administered as a 0.1% w/w injection formulated in 50:50 FCA and water for injection. The middle sites were injected with a 0.1% formulation of the test material in water.
Concentrations: 0.1% w/v solution of copper II sulphate pentahydrate in water for injection or 10% w/w formulation in water for injection and topical
application.
Control group: For the control group the test material was replaced by water in each injection site.
-Topical application:
Test groups: For topical application the test material was prepared as 10% w/w formulation in water for injection and applied as a dose volume of
0.5 mL.
Control group: For the control group the test material was replaced by water.

B. CHALLENGE EXPOSURE
Test material preparation: 0.5 m/L of the test substance was prepared as a 10% (w/w) suspension in water for injection.
Day(s) of challenge: Day 22 of study
Concentrations: 10% w/w solution of copper II sulphate pentahydrate in water for injection was the maximum non-irritant concentration appropriate for use in the challenge phase.
Rechallenge: No

C. SCORING
Reactions were assessed 24 h and 48 h following the challenge application.

D. REMOVAL OF TEST SUBSTANCE
No details provided for any skin washing procedures.
Challenge controls:
All information has been given under 'Details on study design'.
Positive control substance(s):
no
Remarks:
The intradermal injections and topical occlusive application for 48 hours were carried out under the same conditions as in the treated group with water for injection replacing the test substance.

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
10% w/w copper sulphate pentahydrate
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
There were no reactions indicative of skin sensitisation in either the test or control group animals.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10% w/w copper sulphate pentahydrate. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: There were no reactions indicative of skin sensitisation in either the test or control group animals..
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
10% w/w
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
There were no reactions indicative of skin sensitisation in either the test or control group animals.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10% w/w. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: There were no reactions indicative of skin sensitisation in either the test or control group animals..

Any other information on results incl. tables

Pilot test results:

A concentration of 0.1% w/w in water for injection was selected for intradermal injection based on the results of the irritation screen and 10% w/w formulations in water for injection, the maximum non-irritant level, was used for the topical applications.

Study results:

24 hours after challenge:

Incidence index(Number of erythema scores of greater than 0.5 / number of animals evaluated) = 0/20 for test animals and 0/10 for the controls.

Severity index(Sum of erythema scores at 24 hours post challenge / number of animals evaluated) = 0 for test animals and 0 for controls

There were no reactions indicative of skin sensitisation in either the test or control group animals.

48 hours after challenge:

Incidence index(Number of erythema scores of greater than 0.5 / number of animals evaluated) = 0/20 for test animals and 0/10 for the controls

Severity index(Sum of erythema scores at 24 and 48 hours post challenge / number of animals evaluated) = 0 for test animals and 0 for controls

There were no reactions indicative of skin sensitisation in either the test or control group animals.

Overall results:

It was concluded that repeated administration of copper II sulphate pentahydrate did not produce a delayed contact sensitisation response in guinea pigs and is not considered a dermal sensitizer under the study conditions utilised. The historical positive control studies presented n summary with the protocol gave positive responses confirming validity and sensitivity of the methods used in this assay.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information All test animals appeared to be clinically normal throughout the study.
Conclusions:
Copper II sulphate pentahydrate did not produce a delayed contact sensitisation response in guinea pigs and is not considered a dermal sensitizer under the study conditions utilised.
Executive summary:

Materials and Methods:

A test group of twenty Hartley guinea pigs and a control group of ten Hartley guinea pigs were prepared by clipping the hair from the dorsal region. The guinea pigs were observed for clinical signs of reaction to treatment throughout the induction and challenge phases.

During the induction period copper II sulphate pentahydratein aqueous solutionwas applied by intradermal injection in water for injection or Freund’s complete adjuvant and then by topical application a week later. Thenegative control group received the vehicle alone during the induction phase.Twenty-two days after the initial induction application, animals of all groups were challenged by topical application of 0.5 mL in a 10% (w/w) solution in water. Skin reactions were recorded 24 and 48 hours after removal of the dressings following each induction and challenge application. Reactions were graded according to a modified Draizescoring scheme.

Results and discussion:

Challenge phase:- 24 hours after challenge

Incidence index (Number of erythema scores of greater than 0.5 / number of animals evaluated) = 0/20 for test animals and 0/10 for the controls

Severity index (Sum of erythema scores at 24 hours post challenge / number of animals evaluated) = 0 for test animals and 0 for controls.

 

Challenge phase: -48 hours after challenge

Incidence index (Number of erythema scores of greater than 0.5 / number of animals evaluated) = 0/20 for test animals and 0/10 for the controls

Severity index (Sum of erythema scores at 24 and 48 hours post challenge / number of animals evaluated) = 0 for test animals and 0 for controls

All test animals appeared to be normal throughout the study.

Conclusion:

Copper II sulphate pentahydrate did not produce a delayed contact sensitisation response in guinea pigs and is not considered a dermal sensitizer under the study conditions utilised.