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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
Study period:
16 March 1999 to 16 April 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to EU & OECD test guidance in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test

Test material

Constituent 1
Details on test material:
Name: T-9601
Certificate of analysis: NB 203 dated November 16,1998
Purity: 94.7 % (w/w) ± 0.5 %
Appearance: red powder
Melting point: 155°C - 160°C
Solubility: <0.5 g/l in water
Batch number: HI-295
Date of production: May 29, 1998
Date of receiving: July 20, 1998
Date of expiry: May 2001
Storage conditions: darkness at approximately 5°C in a refrigerator
Stability and homogeneity in the vehicle: is guaranteed for 4 hours

In vivo test system

Test animals

Species:
guinea pig
Strain:
Pirbright-Hartley
Sex:
female
Details on test animals and environmental conditions:
Species / sex: Pirbright-White guinea pig / female
Strain: HsdPoc:DH
Origin: HARLAN WINKELMANN Gartenstr, 27 D-33178 Borchen SPF breeding colony
Body weight of the main test group at start of study: mean: 369 g (= 100 %)
Min: 322 g (-12.6%)
Max: 416 g (+ 12.7%)
N: 15
Randomisation: Randomisation scheme 98.075B
Animal maintenance: in fully air-conditioned rooms in macrolon cages (type 4) on soft wood granulate in groups of 5 animals
Room temperature: 20±3°C
Relative humidity: 50±20%
Lighting time: 12 hours daily
Acclimatisation: at least 7 days
Food: ssnlff8 Ms-H (V2233), ad libitum
Water: tap water in plastic bottles, ad libitum
Animal identification: fur marking with KMnO4 and cage numbering

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: sesame oil
Concentration / amount:
Intradermal induction was performed using 5 % T-9601 in sesame oil. Dermal induction and challenge treatment were carried out with 25 % T-9601 in sesame oil.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: sesame oil
Concentration / amount:
Intradermal induction was performed using 5 % T-9601 in sesame oil. Dermal induction and challenge treatment were carried out with 25 % T-9601 in sesame oil.
No. of animals per dose:
Determination of the primary non-irritant concentration – 3 animals
Determination of the tolerance of the intradermal injections – 2 animals
Control group – 5 animals
Treatment group -10 animals
Details on study design:
The following preparations were used for the intradermal injections:

Control group
50 % Freund's Complete Adjuvant emulsion
Original Freund's Complete Adjuvant (Sigma Chemical Company) was mixed immediately before use with an equal volume of deionised water.
Sesame oil (Vehicle)
50 % Freund's Complete Adjuvant emulsion mixed with an equal volume of the vehicle

Treatment group
50 % Freund's Complete Adjuvant emulsion
Original Freund's Complete Adjuvant (Sigma Chemical Company) was mixed immediately before use with an equal volume of deionised water.
5 % T-9601 in sesame oil
5 % T-9601 in a 60 % Freund's Complete Adjuvant emulsion

For the intradermal injections of the test substance in 50 % Freund's adjuvant, T-9601 was suspended in Freund's Original Adjuvant and then mixed with an equal volume of deionised water [percentages w/v].

For the dermal treatments, T-9601 was suspended in sesame oil [percentages w/v].

Determination of the primary non-Irritant concentration
Prior to the determination of the primary non-irritation concentration in a dermal-occlusive test the animals received 4 intradermal injections of a 50% Freund's Complete Adjuvant emulsion (4 x 0.1 ml) into the dorsal area, since Freund's Complete Adjuvant may lower the threshold of primary irritation. Thereafter, each of the following test concentrations was administered to the flanks of two guinea pigs:
25.0 % in sesame oil
5.0 % in sesame oil
1.0 % in sesame oil

The hair on the flanks of the animals was removed mechanically. 0.5 ml of the test substance preparation was administered to a 2 x 2 cm cellulose patch, which was fixed to the flank and covered occlusively for 24 hours with a bandage and film. 24 hours after removal of the patches, the treated skin areas were examined for erythema and edema.

Determination of the tolerance of the intradermal injections
To determine the tolerance of intradermal injections, each of the following prepara¬tions was administered twice by intradermal injection to 2 guinea pigs. The injection sites (sites 1, 2 and 3) were all within a dorsal area measuring 2 x 4 cm in the vicinity of the shoulders.

Site appl.vol. conc. Vehicle
1 2 x 0.1ml 5.0% sesame oil
2 2 x 0.1ml 1.0% sesame oil
3 2 x 0.1ml 0.2% sesame oil

24, 48, 72 and 96 hours after administration the injection sites were examined tor local tolerance.

Main test for the sensitizing properties
Chronological description of the test procedure indicating the day, at which procedure was carried out, on the left margin of the page.

Study day
0 The body weight of the animals was determined.
The guinea pigs were shaved mechanically over a dorsal area of 4 x 6 cm in the vicinity of the shoulders.
1 Intradermal induction treatment
Two intradermal injections per animal of the following preparations. The injection sites (site 1, 2 and 3) were all within a dorsal area of 2 x 4 cm. The injection sites were left uncovered.
Treatment group:
Site appl.vol. conc. Preparation
1 2 x 0.1ml - 50% Freund’s Adjuvant
2 2 x 0.1ml 5% substance in sesame oil
3 2 x 0.1ml 5% substance in 50% Freund’s Adjuvent

Control group:
Site appl.vol. preparation
1 2 x 0.1ml 50% Freund’s Adjuvent
2 2 x 0.1ml sesame oil
3 2 x 0.1ml equal volume of sesame oil and 50% Freund’s Adjuvent

2-7 The administration area was examined for local tolerance. Any systemic toxic effects were recorded.

8 Dermal induction treatment
An amount of 0.5 ml of the test substance preparation (treatment group) or the vehicle (control group) was administered to a 2 x 4 c m cellulose patch. This patch covered the area where the intradermal injection had been placed. The administration area was then kept for 48 hours under an occlusive bandage with an impermeable film and an elastic bandage.

Treatment group: 25.0 % test substance in sesame oil
Control group : sesame oil

10 Occlusive bandage removed, irritant effects recorded.

11-21 No treatment of control or treatment group. Test animals kept under observation.

22 Dermal challenge treatment
One area of approx. 5 x 5 cm on the left flank was shaved mechanically.
An amount of 0.5 ml of the test substance preparation was administered to a 2 x 2 cm cellulose patch. The administration area was t hen kept for 24 hours under an occlusive bandage with an impermeable film and an elastic bandage.

Treatment and control group (left flank); 25.0 % T-S601 in sesame oil

23 Occlusive bandage removed. Any remnants of the test substance were carefully washed off with warm water.

24 Examination of the skin approx. 24 hours after removal of the patches.

25 Examination of the skin approx. 48 hours after removal of the patches. Body weights of the test animals determined.


29 Repeated dermal challenge treatment

One area of approx. 5 x 5 cm on the right flank was shaved mechanically.
An amount of 0.5 ml of the test substance preparation was administered to a 2 x 2 cm cellulose patch. The administration area was then kept for 24 hours under an occlusive bandage with an impermeable film and an elastic bandage.

Treatment and control group (right flank):

25.0 % T-9601 in sesame oil

30 Occlusive bandage removed. Any remnants of the test substance were carefully washed off with warm water.

31 Examination of the skin approx. 24 hours after removal of the patches.

32 Examination of the skin apprax. 48 hours after removal of the patches. Body weights of the test animals determined.
Challenge controls:
Dermal challenge treatment
One area of approx. 5 x 5 cm on the left flank was shaved mechanically.

An amount of 0.5 ml of the test substance preparation was administered to a 2 x 2 cm cellulose patch. The administration area was then kept for 24 hours under an occlusive bandage with an impermeable film and an elastic bandage.

Treatment and control group (left flank); 25.0 % T-S601 in sesame oil
Positive control substance(s):
yes
Remarks:
alpha-hexyl cinnamic

Results and discussion

Positive control results:
Dermal induction was performed using 50 % alpha-hexyl cinnamic aldehyde in polyethylene glycol 400 (Riede! de Haen AG). The control group was exposed to polyethylene glycol 400 only. Challenge treatment was carried out with 25 % alpha-hexyl cinnamic aldehyde in polyethylene glycol 400,

Based on the results of this study alpha-butyl cinnamic aldehyde may cause sensitization by skin contact

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Any other information on results incl. tables

First challenge treatment – control and treatment group

 

25% T-9601 in sesame oil (day 22)

 

Treated area: left flank

 

Scoring for dermal reactions

Time of observation: approx. 24 hours after removal of the patches (day 24)

Control animal No.

1

2

3

4

5

 

 

 

 

 

Value

0

0

0

0

0

 

 

 

 

 

Treated animal No.

6

7

8

9

10

11

12

13

14

15

Value

0

0

0

0

0

0

0

0

0

0

Time of observation: approx. 48 hours after removal of the patches (day 25)

Control animal No.

1

2

3

4

5

 

 

 

 

 

Value

0

0

0

0

0

 

 

 

 

 

Treated animal No.

6

7

8

9

10

11

12

13

14

15

Value

0

0

0

0

0

0

0

0

0

0

After the challenge treatment no animal of the treatment group showed a positive skin reaction during the observation period.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the present study, none of ten animals of the treatment group showed a positive skin response after challenge procedure.
Thus, the percentage of animals reacting positive is below the threshold of 30 %.
Based on the results of this study T-9601 showed no evidence for sensitizing properties.
Executive summary:

Chemical substances may produce an immunological reaction (sensitization) after single or repeated dermal administration. Further administration after the elapse of a certain period may cause allergic reactions. The objective of the test procedure described here is to determine the potential sensitizing properties of a substance.

 

The present study was conducted in compliance with EEC-Guideline B.6 " Sensitization of the Skin" of the Directive 96/54/EEC: Commission Directive of July 30,1998 adapting to technical progress for the twenty-second time Council Directive S7/54S/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances and OECD-Guideline for testing of chemicals, 406 "Skin Sensitization", adopted July 17,1992.This study was conducted in compliance with the Principles of Good Laboratory Practice (GLP).

 

Testing for sensitizing properties of T-9601 was performed in female guinea pigs according to the method of MAGNUSSON & KLIGMAN.

 

Intradermal induction was performed using 5 % T-9601 in sesame oil. Dermal induction and challenge treatment were carried out with 25 % T-9601 in sesame oil. The validity of the test system is confirmed by the periodically conducted positive control test using alpha-hexyl cinnamic for the BUEHLER test (report number 98.1044, dated January 12,1999; Hoechst Marion Roussel, Deutschland GmbH).

 

Based on the results of this study T-9601 showed no evidence for sensitizing properties.

 

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