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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 Authority for Biocides and Existing Substances Regulations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
An LLNA study was not conducted because adequate data from a guinea pig maximisation test are already available. 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:
Lot/batch number: 02-0084
Description: Brown/black powder.
Purity: 97.7% cupric oxide
Stability: Stable at room temperature.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
Source: David Hall Limited, Burton-on-Trent, Staffordshire, UK (intradermal and topical challenge sighting studies and main study) and Harlan UK
Limited, Bicester, Oxon, UK (topical induction sighting study only).
Age/weight at study initiation: The mean bodyweight range was reported to be 300 to 450g and were 8 to 12 weeks old.
Sex: Male animals were used in the main and sighting test studies. Positive control studies were carried out in both male and females.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
arachis oil
Concentration / amount:
Concentrations used for induction:
Intradermal induction: 0.1% w/w in distilled water (causing mild to moderate irritation).
Topical Induction: 75% w/w in distilled water (causing mild to moderate irritation).

Concentrations used for challenge:
Topical challenge: 10% w/w and 5% w/w in arachis oil BP (usually maximum non-irritant concentration).
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
arachis oil
Concentration / amount:
Concentrations used for induction:
Intradermal induction: 0.1% w/w in distilled water (causing mild to moderate irritation).
Topical Induction: 75% w/w in distilled water (causing mild to moderate irritation).

Concentrations used for challenge:
Topical challenge: 10% w/w and 5% w/w in arachis oil BP (usually maximum non-irritant concentration).
No. of animals per dose:
Number of animals per group: 10 test animals were used in the main study.
Details on study design:
Pretest performed on irritant effects: Yes, sighting tests were carried out to determine the concentration of test material to be used at each stage of
the main study.

Induction Schedule: Day 0 – day 7

On day 0, an area of 40 mm x 60 mm of hair was clipped from each animal using veterinary clippers and three pairs of 0.1 ml intradermal injections
were made on either side of the mid-line. The injections were:

a) Freund’s Complete Adjuvant plus distilled water at a ratio of 1:1
b) 0.1 % w/w formulation of the test material in arachis oil BP
c) 0.1 % w/w formulation of the test material in a 1:1 preparation of Freund’s Complete Adjuvant plus distilled water.

Approximately 24 and 48 hours later, the degree of erythema at the test material injection sites (injection b) was evaluated.

On day 7, the same area was clipped again on each animal and treated with a topical application of test material (75 % w/w in arachis oil BP) and held in place with occlusive dressing for 48 hours. After 1 and 24 hours, the degree of erythema and oedema was evaluated after removal of the dressings.

Induction of the control animals was performed in an identical manner as for the test animals, except that the test material was ommitted.

The scoring schedule for erythema was derived from 'Modified OECD Test Guideline 406, 1992 and Method B6 Skin Sensitisation of Commission
Directive 96/54/EEC' and the scoring schedule for oedema was taken from Draize, J.H. 1977.

See Table 1.

Way of induction: Intradermal and topical. Topical induction was kept in place with an occlusive dressing.


Challenge Schedule: Day 21; see Table 1.

On day 21, an area of 50 mm x 70 mm on both flanks was clipped free of hair and a filter paper patch loaded with test material at the maximum
non-irritant concentration (10 % w/w in arachis oil BP) was applied to the right flank of each animal and held in place with surgical tape and an
occlusive dressing. To ensure the maximum non-irritant concentration was used at challenge, the test material was applied in a similar method to the left flank at a concentration of 5 % w/w in arachis oil BP.

The dressings were kept in place for 24 hours, after which the dressing was removed and the challenge sites swabbed with cotton wool soaked in diethyl ether to remove residual material. Approximately 24 and 48 hours after challenge dressing removal, the degree of erythema and
oedema was evaluated. Any other reactions were also recorded.
Challenge controls:
5 negative control animals were used in the main study.
Positive control substance(s):
yes
Remarks:
2-mercaptobenzothiazole and alpha-Hexylcinnamaldehyde, both 5% in arachis oil BP

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
10%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Discrete or patchy erythema was seen at the topical challenge sites of 4 test group animals at the 24 hour observation.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Discrete or patchy erythema was seen at the topical challenge sites of 4 test group animals at the 24 hour observation..
Reading:
2nd reading
Hours after challenge:
24
Group:
test group
Dose level:
5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Discrete or patchy erythema was seen at the topical challenge sites of 2 test group animals at the 24 hour observation.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 24.0. Group: test group. Dose level: 5%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Discrete or patchy erythema was seen at the topical challenge sites of 2 test group animals at the 24 hour observation..
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
10%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No reactions were observed at the 48 hour observation.
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No reactions were observed at the 48 hour observation..
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No reactions were observed at the 48 hour observation.
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 5%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No reactions were observed at the 48 hour observation..

Any other information on results incl. tables

Results of Test

The results are summarised in Tables 1 and 2 below.

24 h after challenge:

10% w/w challenge concentration: 4/10 test animals showed signs of discrete or patchy erythema and 0/10 test animals showed signs of oedema after 10% w/w test material challenge. 8/10 test animals and 3/5 control animals showed black/grey coloured staining.

5% w/w challenge concentration: 2/10 test animals showed signs of discrete or patchy erythema and 0/10 test animals showed signs of oedema after 5% w/w test material challenge. 1/10 test animals and 1/5 control animals showed black/grey coloured staining.

0/5 control animals showed signs of erythema or oedema at either the 10% or 5% w/w challenge concentration after 24 hours.

48 h after challenge:

10% w/w challenge concentration: 0/10 test animals showed signs of erythema and 0/10 test animals showed signs of oedema after 10% w/w test material challenge. 7/10 test animals and 3/5 control animals showed black/grey coloured staining.

5% w/w challenge concentration: 0/10 animals showed signs of discrete or patchy erythema and 0/10 showed signs of oedema after 5% w/w test material challenge. 1/10 test animals and 1/5 control animals showed black/grey coloured staining.

0/5 control animals showed signs of erythema or oedema at either the 10% or 5% w/w challenge concentration after 48 hours.

Table 1. Detailed information including induction/challenge/scoring schedule for sensitisation test.

 

INDUCTION/

CHALLENGE

DAY OF

TREATMENT

APPLICATION

OBSERVATIONS/REMARKS

Induction 1

 

Intradermal injection

0

3 intradermal injections made as follows;

·         FCA & distilled water in ratio 1:1

·         0.1% w/w formulation of the test material in arachis oil

·         0.1% formulation of the test material in a 1:1 preparation of FCA plus water.

Degree of erythema quantified at 24 and 48 hours following injections.

Moderate and confluent erythema was noted at the intradermal induction sites of test animals.

 

Discrete or patchy erythema was noted at the intradermal sites of the control group animals.

Pre-treatment for non irritating substance

There was no pre-treatment for non irritating substance

Induction 2

 

Topical induction

 

7

Filter paper with 75% test material in arachis oil applied to skin for 48 hours. Degree of erythema and oedema quantified 1 and 48 hours following removal of patch.

Black staining, which prevented evaluation of erythema, was noted at the topical induction sites of all test animals at the 1 and 24 hour observations.

 

Discrete or patchy erythema was noted at the topical induction sites of the control group animals.

 

Bleeding from the intradermal injection sites was noted in five test group animals and three control group animals.

 

Challenge

 

Topical challenge

21

Filter paper with 10 % test material in arachis oil applied to each animal. To ensure a maximum non-irritant concentration was used at challenge, the test material was also applied at 5% w/w in arachis oil.

 

The test material was removed after 24 hours. After 24 and 48 hours following challenge dressing removal, the degree of erythema and oedema was quantified.

Black/grey coloured staining was noted at the topical challenge sites of up to 8 test and 3 control group animals. The staining did not affect evaluation of skin responses.

 

10% w/w in arachis oil

 

Transient challenge reactions (Discrete or patchy erythema) were noted at the topical challenge sites of four test group animals at the 24-hour observation. These reactions were not apparent at the 48- hour observation and were therefore not attributed to contact sensitisation.

 

No skin reactions were noted at the challenge sites of the control group animals at the 24 or 48 hour observations

 

5% w/w in arachis oil

 

Transient challenge reactions (discrete or patchy erythema) were noted at the topical challenge sites of two test group animals at the 24-hour observation. These reactions were not apparent at the 48-hour observation and were therefore not attributed to contact sensitisation.

 

No skin reactions were noted at the challenge sites of the control group animals at the 24 or 48 hour observations.

FCA – Freund’s Complete Adjuvant

Table 2. Results of skin sensitisation test.

 

 

Number of animals with signs of allergic reactions

/number of animals in group

Negative Control

Test Group

Positive Control

5%

10%

(1)

(2)

Scored after 24-hours

0/10

2/10*

4/10*

-

-

Scored after 48-hours

0/10

0/10

0/10

10/10

10/10

9/9

5/10

4/10

2/10

(1) 2-mercaptobenzothiazole (3 studies)

(2) α-Hexylcinnamaldehyde (3 studies)

 

*As these reactions were not present at 48-hour observation time point, they were not considered to be attributed to contact sensitisation.

Overall result

Results after intradermal and topical induction:

Moderate and confluent erythema was seen at all the intradermal induction sites of the test animals at the 24 and 48 hour observation time points. In comparison, discrete or patchy erythema was noted at all the intradermal induction sites of the control group animals after 24 hours and at 3/10 sites after 48 hours.

After topical induction, black staining prevented the evaluation of erythema in all the test group animals, while 0/10 test animals showed signs of oedema, at the 1 and 24 hour observation time points. Discrete or patchy erythema was noted in all the control animals at 1 hour, which was reversed at the 24 hour observation time point. Bleeding was noted in 5/10 test animals and 3/5 control animals after topical induction.

Results after topical challenge:

After topical challenge, black/grey staining was noted in test and control animals exposed to both 10% and 5% w/w test material concentration in arachis oil. This staining did not affect evaluation of skin responses.

The discrete or patchy erythema noted after 24 hours in both the 10% and 5% w/w concentration sites disappeared after 48 hours . These reactions were, therefore, not associated with contact sensitisation.

No skin reactions were noted at the challenge sites of the control animals at the 24 or 48-hour observations at the 10% or 5% w/w concentrations.

Results of Pilot Studies

Intradermal induction sighting test:

The highest concentration causing only mild to moderate skin irritation which was well tolerated systemically (0.1% w/w) was selected for the intradermal induction stage of the main study.

Topical induction sighting test:

The highest concentration applied causing only mild to moderate dermal irritation which was well tolerated systemically (75% w/w) was selected for the topical induction stage of the main study.

Topical challenge sighting test:

The highest non irritant concentration of the test material and one lower concentration were selected for the topical challenge stage of the main study (10% and 5% w/w in arachis oil BP).

Other findings:

Three out of the four animals given test material at concentrations of 0.5, 1 and 5% w/w in the intradermal induction sighting test were humanely killed due to the severity of reactions.

One animal in the topical induction sighting test was found dead approximately 48 hours after dosing. The cause of death was not determined but was considered not to be related to the toxicity of the test material.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Under the conditions of the test, the test material produced a 0% (0/10) sensitisation rate and was classified as a non-sensitiser to guinea pig skin.
The test material did not meet the criteria for classification as a sensitiser according to EU labelling regulations Commission Directive 93/21/EEC.
Executive summary:

Materials and Methods

The study was performed to assess the contact sensitisation potential of copper oxide in the albino guinea pig. Ten test and five control animals were used for the study. Two phases were involved; an induction of a response by intradermal injection and topical application, and a topical challenge of that response. Based on the results of sighting tests, the concentrations of the test material for the induction and challenge phases were selected as;

Intradermal induction: 0.1% w/w in distilled water

Topical Induction: 75% w/w in distilled water

Topical challenge: 10 and 5% w/w in distilled water

 

On day 0, approximately 24 and 48 hours after the initial intradermal induction injection (0.1% w/w), the degree of erythema was evaluated. Seven days later, the same area used for the intradermal injection was treated with a topical application of test material (75% w/w). The degree of erythema and oedema was evaluated 1 and 24 hours after removal of the patches. Induction of the control animals was performed in an identical manner as for the test animals, except that the test material was ommitted.

 

On day 21, test material was applied at the maximum non-irritant concentration (10% w/w) and a lower concentration (5% w/w) as challenge doses. Approximately 24 and 48 hours after removal of the challenge doses, the degree of erythema and oedema was evaluated and any other skin reactions were recorded.

 

The study was conducted according to Commission Directive 96/54/EC Method B6 Acute Toxicity (Skin Sensitisation) and OECD Guidelines for the Testing of Chemicals No. 406 'Skin Sensitisation' (adopted 17 July 1992). The study was also conducted according to GLP.

 

No deviations from the test guidelines, or deficiencies in the method were reported. Results and Discussion Discrete or patchy erythema was noted in 4/10 and 2/10 animals challenged with test material after 24 hours, at concentrations of 10% w/w and 5% w/w respectively. In all cases, the erythema disappeared after 48 hours. These reactions were, therefore, not associated with contact sensitisation.