REAKTIV-ORANGE DYPR 934

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999-03-02-1999-04-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted in compliance with EEC-Guideline B6 and OECD Guidelines for testing chemicals 406 according to the good laboratory practice regulations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Test material

Test material information

Constituent 1

Details on test material:

-Name of test material (as cited in study report): Reaktiv-Orange DYPR934

In vivo test system

Test animals

Species:

guinea pig

Strain:

Pirbright-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Species: Pirbright-white guinea pig/female
-Strain: Hsd Poc:DH

-origin: Harlan Winkelmann, Gartenstr. 27 D-33178 Borchen (SPF breeding colony)
- Weight at study initiation: mean = 426 g (= 100 %)
min = 404 g (-5.2 %)
max: = 454 g(+6.6 %)
n = 15
Randomization: Randomization scheme 98.0753
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum
- Acclimation period: at least 7 days
-Food: Ssniff® Ms-H (V2233) ad libitum.
ENVIRONMENTAL CONDITIONS
- Room temperature (°C):20 ± 3 °C
- Humidity (%): 50 ± 20 %
- Photoperiod (hrs dark / hrs light): 12 hours daily
Maintenance: in a fully air conditioned room in macrolon cages granulate (type 4) on soft wood granulate in groups 5 animals.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

water

Remarks:

deionized water.

Induction 2

Concentration / amount:

Concentration of test material and vehicle used for each exposure phase:

Intradermal induction:
-5 % test substance in deionized water
-Control group: 0 %
Dermal induction:
Treatment group: 25 % test substance in deionized water
Control group: deionized water
Dermal challenge.
Treatment group and control group (first and second reading): 25.0 % Reaktiv-Orange DYPR 934 in deionized water

25 % deionized water

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

water

Remarks:

deionized water.

Challenge 2

Concentration / amount:

Concentration of test material and vehicle used for each exposure phase:

Intradermal induction:
-5 % test substance in deionized water
-Control group: 0 %
Dermal induction:
Treatment group: 25 % test substance in deionized water
Control group: deionized water
Dermal challenge.
Treatment group and control group (first and second reading): 25.0 % Reaktiv-Orange DYPR 934 in deionized water

25 % deionized water

No. of animals per dose:

Number of animals in test group: 10 Number of animals in negative control group: 5

Details on study design:

RANGE FINDING TESTS:

MAIN STUDY
A. INDUCTION EXPOSURE
CHROLOGICAL DESCRIPTION OF THE TEST PROCEDURE
STUDY DAY TEST PROCEDURE
0 The body weight of the animals was determined.
The guinea pigs were shaved mechanically over a dorsal area of 4X 6 cm in the vicinity of the shoulders.

1 INTRADERMAL INDUCTION TREATMENT
Two intradermal injections per animal of the following preparations.
The injection site (1,2,3) were all within a dorsal area of 2x4 cm. The injection sites were left uncovered.

Treatment group:
site appl. vol conc. preparation
1 2x0.1 mL - 50 % Freund's Adjuvant
2 2x0.1 mL 5 % substance in deionized water
3 2x0.1 mL 5 % substance in 50 % Freund's Adjuvant

Control group:
site appl. vol conc preparation
1 2x0.1 mL - 50 % Freund's Adjuvant
2 2x0.1 mL 5 % deionized water
3 2x0.1 mL 5 % equal volume of deionized water and 50 % Freund's Adjuvant

2-7 The administration area was examined for local tolerance. Any systemic toxic effects were recorded.
8 DERMAL INDUCTION
An amount of 0.5 mL of the test substance preparation (treatment group) or the vehicle (control group) was administrated to 2x 4 cm 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 elastic bandage.
Treatment group: 25 % test substance in deionized water.
Control group: deionized water.

10 Occlusive bandage removed, irritant effects recorded.

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

B. CHALLENGE EXPOSURE

22 An amount of 0.5 mL of the test substance preparation was administrated to a 2x2 cm cellulose patch. The administration area was then kept for24 hours under an occlusive bandage with an impermeable film and elastic bandage.
Treatment and control group (left flank)

25.0% Reaktiv-Orange DYPR 934 in deionized water.

23 Occlusive bandage removed. Any remnants of the test substance were carefully washed off with warm water.
24 Examination of the skin approx. 24 hr after removal of the patches.
25 Examination of the skin approx. 48 hr after removal of the patches. Body weight of the test animal determined.

29 Repeat dermal treatment
Treatment and control group (right flank)
25.0 % Reaktiv-Orange DYPR 934 in deionized water.
Handling analogous to the first challenge treatment
30 Occlusive bandage removed. Any remnants of the test substance were carefully washed off with warm water.

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

32 Examination of the skin approx. 48 hr after removal of the patches.
Body weight of the test animals determined.


- Evaluation (hr after challenge):
Erytheme and edema are major clinical indicators of an allergic reaction. The decisive criterion for evaluation of the sensitizing properties of a test substance is the number of sensitized test animals, not the intensity of the dermal reaction.
The substance is considered to be sensitizing if 30 % more of animals in the treatment group definitely showed a positive skin reaction and at the same time nor irritant effects have emerged in the control group.

Positive control substance(s):

yes

Remarks:

The validity of the test system is confirmed by the periodically conducted positive control using cinnamic aldehyde (CAS No. 101-86-0) for Buehler-Test (report No.:98.1044; 1999-01-12; Hoechst Marion Roussel, Deutschland GmbH)

Results and discussion

Positive control results:

See comments below

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 3

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 4

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 5

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.

Results 6

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.

Results 7

Reading:

rechallenge

Hours after challenge:

24

Group:

negative control

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 24.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 5.0.

Results 8

Reading:

rechallenge

Hours after challenge:

48

Group:

negative control

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 5.0.

Any other information on results incl. tables

 

Scale for scoring dermal reactions According to the scale of EEC-Guideline B.6: Magnusson/Kligmann grading Scale for the evaluation of challenge patch test reactions:
No visible change	0
Discrete or patchy erythema	1
Moderate and confluent erythema	2
Intense erythema and swelling	3

 

First challenge treatment-Control and treatment group 25% Reaktiv-Orange DYPR 934 in deionized water (day 22) Treatment area: left flank
Scoring of dermal reactions Time 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 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
Second challenge treatment-Control and treatment group 25% Reaktiv-Orange DYPR 934 in deionized water (day 29) Treatment area: right flank
Scoring of dermal reactions Time observation: approx. 24 hours after removal of the patches (day 31)
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 observation: approx. 48 hours after removal of the patches (day 32)
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 30 of the study.}

 

 

                                                  

Table of individual data Determination of the primary non irritant concentration –Individual values Treated area: left and right flank Time of observation: approx. 24 hours after removal of the patches
Animal No.:	Concentration [%]	Results
1 left flank	25	0
1 right flank	5	0
2 left flank	25	0
2 right flank	1	0
3 left flank	5	0
3 right flank	1	0
Determination of tolerance of the intradermal injections-Individual values
Time after injection	Findings
	5%	1%	0.2%
Day 1	Slight erythema and edema	Slight erythema	no findings
Day 2	Slight erythema	no findings	no findings
Day 3	Slight erythema	no findings	no findings
Day 4	Slight erythema	no findings	no findings

 

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

The intradermal injections with Freund's Ajuvant 

(with and without test substance) caused severe erythema and eodema as well as indurations and encrustations.

Dermal induction treatment:

After removal of the patches at day 10, severe erythema and
edema, indurated and encrusted skin as well as necrosis were
observed at the sites previously treatd with Freund's
Adjuvant.

The administration sites treated with the test substance
alone showed no signs of irritaion.

Evidence of sensitization of each challenge concentration:

Treatment group:   0/9

Control group:    0/5
Other observations:
One animal of the treatment group was found dead at

day 30 of the study.

No toxic signs were observed.

None of nine animals of the treatment group showed

signs of intoxication throughout the study.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information REAKTIV-ORANGE DYPR 934 showed no evidence of sensitizing properties. Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of the 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 934 showed no evidence of sensitizing properties.

Executive summary:

Testing for sensitizing properties of Reaktiv-Orange 934 DYPR was performed in (nulliparous and non-pregnant) female guinea pig according to the method of Mangusson & Kligman.

Based on the fact that, no signs of irritation occured after administration of the different test concentrations, a concentration of 25 % Reaktiv-Orange DYPR 934 in deionized water was chosen for dermal as well as for challenge induction.

A 5 % preparation was selected for the intradermal injections in the main test.

The Reliability check of the test system is confirmed by the periodically conducted positive control using cinnamic aldehyde (CAS No. 101-86-0) for Buehler-Test (report No.:98.1044; 1999-01-12; Hoechst Marion Roussel, Deutschland GmbH).

Under the conditions of the 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 934 showed no evidence of sensitizing properties.

 

Results Synopsis:

Test substance	Objective of the study	Test animals/strain	Test conditions	Reliability check	vehicle	Results/findings
Reaktiv-Orange DYPR 934	Determination of the potential sensitizing properties of Reaktiv-Orange DYPR 934	15 (nulliparous and non-pregnant) guinea pig, Pirbright/ White Hsd Poc:DH	The hair of the flank was removed mechanically -0.5 mL of the test substance was administrated to a cellulose patch fixed on the flank and covered occlusively -ENVIRONMENTAL CONDITIONS - Temperature ( °C): 20+/- 3 °C - Humidity (%): 50 +/- 20 % - Photoperiod (hrs dark / hrs light): 12 hours daily	The validity of the test system is confirmed by the periodically conducted positive control using cinnamic aldehyde (CAS No. 101-86-0) for Buehler-Test (report No.:98.1044; 1999-01-12; Hoechst Marion Roussel, Deutschland GmbH)	Vehicle: deionized water	-No signs of irritation occurred after administration of different test concentrations. -Body weight gain of the animals was not impaired. -One animal of the treatment group was found dead at day 30 of the study. But this incident was not related to the substance.