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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: GLP and appropriate guidelines
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Principles of method if other than guideline:
not relevant
GLP compliance:
yes
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
E-AF098T (Lot # 474-0306-17), a white powder. Stored at room temp, dry, closed container.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
Source - Harlan, Italy

Number of animals - Screening tests: Intradermal injection tolerance test: 1 group of 2 animals. Topical application tolerance test: 1 group of 5 animals. Main study: 1 control group of 10 animals and 1 test group of 20 animals.
Age of start of treatment- Approximately 4-5 weeks old.
Bodyweight: 250 to 300 g
Husbandary: Temperature range: 22oC ± 2. Relative humidity range: 55± 15%, respectively.
Light: 12 hr artificial light 12 hr dark.
Accommodation: Up to 10 animals/cage during the acclimization period and up to 5 animals/cage during the study.
Acclimatisation period was at least 5 days before start of the treatment
Diet- 8GP17 (Mucedola S.r.i.), ad libitum
Water-Suplied ad libitum to each cage via a water bottle.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Concentration / amount:
Concentration of test material and vehicle used at induction: First Induction- intradermal: 5% test item in vehicle (corn oil). Second induction- topical application- 50% in vehicle (corn oil).
Dose volume: 0.4 ml of test material/animal

Treatment of the test group: test item, 50% in corn oil

Concentration of test material and vehicle used for each challenge:
Dose volume: 0.2 ml of test material/each site

Treatment of the right flank (test and control groups): test
item, 50% in corn oil
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
Concentration of test material and vehicle used at induction: First Induction- intradermal: 5% test item in vehicle (corn oil). Second induction- topical application- 50% in vehicle (corn oil).
Dose volume: 0.4 ml of test material/animal

Treatment of the test group: test item, 50% in corn oil

Concentration of test material and vehicle used for each challenge:
Dose volume: 0.2 ml of test material/each site

Treatment of the right flank (test and control groups): test
item, 50% in corn oil
No. of animals per dose:
Main study:
Number of animals in test group: 20
Number of animals in negative control group: 10
Details on study design:
Selection of suitable concentrations for the main test study:
The study was devided into 2 distinct phases. The first was a dose-rangeing screen which was used to determine suitable dose levels for use in the second phase. This second phase corresponded to the main study, a determination of the sensitization potential of the test item.

RANGE FINDING TEST:
Based on results of sightsing tests, the concentrations of test material for induction and challenge phases were selected as follows:
a) Intradermal induction: 5% in corn oil and 5% in the Emulsified Freund's complete adjuvant.
Topical induction: 50% in corn oil
b) Topical challenge: 50% in corn oil

MAIN STUDY
The control group was composed of 10 animals. The test group was composed of 20 animals.
Two phases were involved: 1)an induction of response and 2) a challenge of that response.

1) Induction of the test animals:
Shortly before tratment the hair was removed from an area of 20mm x 40mm. 3 pairs of sites were selected for each animal. All injections were made
at the edge of the prepared site.
A row of 3 injection (0.1 ml of test material/each site) was given to each animal:
a) Emulsified Freund's complete adjuvant to the animal's anterior site
b) Test item at 5% w/v concentration in the vehicle (corn oil) to the animal's median site
c) Test item at 5% w/v concentration in emulsified Freund's complete adjuvant to the animal's posterior site
Approximately 24 hours after interdermal injection the degree of erythema at the injection site was evaluated (Draize scoring scale).
One week after injection, the same area was treated with a topical application (0.4 ml) of 50% formulation of the test material in corn oil for 48 hours.
The degree of erythema and oedema was quantified 24 hours following removal of the patches.

Induction of the control animals:
A row of 3 injection was given to each animal:
d) Emulsified Freund's complete adjuvant to the animal's anterior site
e) Vehicle (corn oil) to the animal's median site
f) Vehicle mixed with emulsified Freund's complete adjuvant to the animal's posterior site
Approximately 24 hours after interdermal injection the degree of erythema at injection site was evaluated. One week after injection, the same area was treated with a topical application of the vehicle (corn oil) following the same procedure as for the test animals.

2) Challenge
On day 22, 0.2 ml of 50% w/v test substance in corn oil (maximum non- irritant concentration) was applied to the right flank of each animal (test and control groups) and 0.2 ml corn oil was applied to the left flank (test and control groups) for 24 hours. Assessment for skin reactions were performed after a further 24 and 48 hours (24 and 48 hours after challenge dressing removal).
The degree of erythema at injection site was quantified using 'Draize' scale.

The precentage of animals that showed a more sever reaction in the test material challenge site than the most severe reaction seen in the control animals was compared with a scale.



Challenge controls:
Two weeks after the second induction stage, all animals (both treated and control groups) were challenged by topical application of both the vehicle (corn oil) and the test item at 50% concentration.
Positive control substance(s):
no

Results and discussion

Positive control results:
not relevant

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No deaths occured . No remarkable changes in body weight. No other observations were performed
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50 %. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No deaths occured . No remarkable changes in body weight. No other observations were performed.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No deaths occured . No remarkable changes in body weights. No other observations were performed.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50 %. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No deaths occured . No remarkable changes in body weights. No other observations were performed..
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No deaths occured . No remarkable changes in body weights. No other observations were performed.
Remarks on result:
other: see Remark
Remarks:
Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 50 %. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No deaths occured . No remarkable changes in body weights. No other observations were performed..
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No deaths occured . No remarkable changes in body weights. No other observations were performed.
Remarks on result:
other: see Remark
Remarks:
Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 50 %. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No deaths occured . No remarkable changes in body weights. No other observations were performed..
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No deaths occured . No remarkable changes in body weights. No other observations were performed.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No deaths occured . No remarkable changes in body weights. No other observations were performed..
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No deaths occured . No remarkable changes in body weights. No other observations were performed.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No deaths occured . No remarkable changes in body weights. No other observations were performed..
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No deaths occured . No remarkable changes in body weights. No other observations were performed.
Remarks on result:
other: see Remark
Remarks:
Reading: rechallenge. . Hours after challenge: 24.0. Group: negative control. Dose level: 0 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No deaths occured . No remarkable changes in body weights. No other observations were performed..
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No deaths occured . No remarkable changes in body weights. No other observations were performed.
Remarks on result:
other: see Remark
Remarks:
Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. Dose level: 0 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No deaths occured . No remarkable changes in body weights. No other observations were performed..

Any other information on results incl. tables

Preliminary tolerance tests:

No irritation was observed at preliminary topical induction. Therefore, the test item at 50% concentration was used for the topical induction as it was judged to be well tolerated by the test system. The same concentration was judged to be non irritant and was selected for use at challenge.

Induction:
Moderate to severe erythema (score of 3) was apparent at the
sites of intradermal injection following administration of
Freund's complete adjuvant in test and control groups
respectively (anterior sites) and at sites treated with
Freund's complete adjuvant mixed with the test item or
vehicle alone (test and control group, posterior sites).

Very slight erythema (score of 1) was observed at sites
treated with the vehicle alone (control group, median
sites). Very slight to well defined erythema (scores of 1
and 2) was observed at sites treated with the test item
(test group, median sites).


No erythema was observed at sites treated with either
the test item at 50% concentration or the vehicle alone
following 48 hours of topical exposure in animals of the
test and control groups. Hardening of the treated skin sites
was seen in all animals of both groups. This reaction was
attributed to the treatment with Sodium Lauryl Sulphate,
enhanced by the subsequent treatment with the test item (or
by corn oil alone).

Challenge:
No response to the test item at 50% was observed in either
test or control group animals, 24 and 48 hours following a
24 hour topical exposure period. No reaction was observed to
the vehicle alone.

Other observations:
Body weight changes were not remarkable.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information not classified
Conclusions:
Test results indicate that the test item does not elicit a sensitization response in the guinae pigs, being there no evidence of response at challenge following a period of induction exposure to the test item.
Executive summary:

Test results indicate that the test item does not elicit a sensitization response in the guinae pigs, being there no evidence of response at challenge following a period of induction exposure to the test item.