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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:
17 November 1998 to 25 December 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to recent EU & OECD test guidelines 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 of test material (as cited in study report): Reaktiv-Orange DYPR 1410
- Physical state: Orange powder
- Analytical purity: 72%
- Purity test date: December 15, 1998
- Lot/batch No.: DYPR 1410
- Expiration date of the lot/batch: August 2003
- Storage condition of test material: darkness at approximately 5C in a refrigerator

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: HsdPocDH
Origin: HARLAN WINKELMANN Gartenstr. 27 D-33178 Borchen SPF breeding colony
Body weight at start of study: mean = 391g (=100 %)
min = 329g (-15.6%)
max = 417g (+6.7%)
n = 15
Randomization: Randomization scheme 98.0744
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
Acclimatization: at least 7 days

Food: ssniff® 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
Vehicle:
water
Concentration / amount:
Treatment Group:
Appl.vol. Conc. Vehicle
2 x 0.1ml - 50% Freund’s Adjuvent
2 x 0.1ml 5% Substance in deionised water
2 x 0.1ml 5% Substance in 50% Freund’s Adjuvent
Control Group:
Appl. Vol. Vehicle
2 x 0.1ml 50% Freund’s Adjuvent
2 x 0.1ml Deionised water
2 x 0.1ml Equal volume of deionised water and 50% Freund’s Adjuvant
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Treatment Group:
Appl.vol. Conc. Vehicle
2 x 0.1ml - 50% Freund’s Adjuvent
2 x 0.1ml 5% Substance in deionised water
2 x 0.1ml 5% Substance in 50% Freund’s Adjuvent
Control Group:
Appl. Vol. Vehicle
2 x 0.1ml 50% Freund’s Adjuvent
2 x 0.1ml Deionised water
2 x 0.1ml Equal volume of deionised water and 50% Freund’s Adjuvant
No. of animals per dose:
Test group Number of animals
Determination of the primary non-irritant concentration 3
Determination of the tolerance of the intradermal injections 2
Control group 5
Treatment group 10

10 animals in the treatment group and 5 animals in the control group were used. One animal of the treatment group were found dead at day 10 of the study.
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 deionized water.
Deionized water
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 deionized water.
5 % Reaktiv-Orange DYPR 1410 in deionized water
5 % Reaktiv-Orange DYPR 1410 in a 50 % Freund's Complete Adjuvant emulsion

For the intradermal injections of the test substance in 50 % Freund's adjuvant, Reaktiv-Orange DYPR 1410 was dissolved in deionized water and then mixed with an equal volume of Freund's Original Adjuvant [percentages w/v].
For the dermal treatments, Reaktiv-Orange DYPR 1410 was dissolved in deionized water [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 three guinea pigs:

25.0 % in deionized water
5.0 % in deionized water
1.0 % in deionized water

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 oedema.


Determination of the tolerance of the intradermal injections
To determine the tolerance of intradermal injections, each of the following preparations 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.
24, 48, 72 and 96 hours after administration the injection sites were examined for local tolerance.

Challenge controls:
Control Group:
Appl. Vol. Vehicle
2 x 0.1ml 50% Freund’s Adjuvent
2 x 0.1ml Deionised water
2 x 0.1ml Equal volume of deionised water and 50% Freund’s Adjuvant
Positive control substance(s):
yes
Remarks:
Benzocain; periodically conducted positive control test

Study design: in vivo (LLNA)

Vehicle:
other: Not applicable

Results and discussion

Positive control results:
Testing for sensitizing properties of Benzocain was performed in female guinea pigs according to the method of MAGNUSSON & KLIGMAN. This study is conducted periodically, and used as supporting evidence to the fact that the study methodology and approach is appropriate.
Intradermal induction was performed using 1 % Benzocain in sesame oil DAB 10. Dermal induction and challenge treatment were carried out with 25 % Benzocain in sesame oil DAB 10.

After the second challenge treatment four animals of the treatment group (40 %) showed a positive reaction during the observation period. The results obtained are typical for weak sensitizers.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 25%. 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:
9
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: 9.0.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
9
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 9.0.
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: negative control. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
9
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 9.0.
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
9
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 9.0.

In vivo (LLNA)

Results
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Not applicable

Any other information on results incl. tables

First challenge treatment – control and treatment group

25% Reaktiv-Orange DYPR 1410 in deionised water

Treated area; left flank

 

Scoring of dermal reactions

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

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

Time of observation: approx. 48 hours after removal of the patches

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

*Animal was found dead at day 10 of the study.

 

Second challenge treatment – control and treatment group

25% Reaktiv-Orange DYPR 1410 in deionised water

Treated area; left flank

 

Scoring of dermal reactions

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

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

Time of observation: approx. 48 hours after removal of the patches

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

*Animal was found dead at day 10 of the study.

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 nine animals of the treatment group showed a positive skin response after the challenge procedure.

Thus, the percentage of animals reacting positive is below the threshold of 30 %.

Based on the results of this study Reaktiv-Orange DYPR 1410 showed no evidence for sensitizing properties
Executive summary:

The present study was conducted in compliance with EEC-Guideline B.6 "Acute Toxicity Sensitization of the Skin" of the Directive 96/54/EEC and OECD-Guideline for testing of chemicals, 406 "Skin Sensitization".

 

This study was conducted in compliance with GLP.

 

Testing for sensitizing properties of Reaktiv-Orange DYPR 1410 was performed in female guinea pigs according to the method of MAGNUSSON & KLIGMAN.

 

Intradermal induction was performed using 5 % Reaktiv-Orange DYPR 1410 in deionized water. Dermal induction and challenge treatments were carried out with 25 % Reaktiv-Orange DYPR 1410 in deionized water.

 

The validity of the test system is confirmed by the periodically conducted positive control test using benzocain for the maximization test (report number 98.0490, dated June 30, 1998; Hoechst Marion Roussel, Preclinical Development Germany, Drug Safety).

 

One animal of the treatment group was found dead at day 10 of the study. But no substance related toxic effects was noted.

 

Under the conditions of the present study, none of nine animals of the treatment group showed a positive skin response after the challenge procedure.

 

Thus, the percentage of animals reacting positive is below the threshold of 30 %.

 

Based on the results of this study Reaktiv-Orange DYPR 1410 showed no evidence for sensitizing properties.