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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: An apparently well conducted GLP study according to OECD 406 test guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
test commissioned before adoption of LLNA method

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
In the Study Report, the test material, Trigonox 44B, a clear colorless liquid, was said to be identified by the Batch No. 0419906045251. The purity of the test material was stated to be 4.1% active oxygen. The identity, strength and the stability under storage conditions were the responsibility of the Sponsor. Stability or achieved concentration of the test material in the vehicle were not assessed. Certificate of Analysis was not included.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Himalayan
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Supplied by BRL Limited, Basel, Switzerland.
- Age at study initiation: Approximately 4 - 9 weeks old.
- Body Weight: Approximately 500 gms.
- Housing: Five animals per metal cage with wire-mesh floors.
- Diet (e.g. ad libitum): standard guinea pig diet, including ascorbic acid (1000 mg/kg); (Charles River Breeding and Maintenance Diet for guinea pigs, Altromin, Lage, Germany.)
- Water (e.g. ad libitum): free access to tap water
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 degrees Celcius
- Humidity (%): 30 - 70%
- Air changes (per hr): 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
water
Concentration / amount:
Main study: Intradermal injection with a 5% concentration and epidermal challenge with a 100% concentration.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Main study: Intradermal injection with a 5% concentration and epidermal challenge with a 100% concentration.
No. of animals per dose:
10 females in the experimental group; 5 females in the control group.
Details on study design:
PRELIMINARY IRRITATION STUDY
A preliminary irritation study was conducted in order to select test substance concentrations to be used in the main Study. The selection of concentrations was based on the following criteria:

- The concentrations are well-tolerated by the animals.
- For the induction exposures: the highest possible concentration that produced mild to moderate irritation (grades 2 - 3).
- For challenge exposure: the maximum non-irritant concentration.

Series of test substance concentrations were tested. Practical feasibility of administration determined the highest starting-concentration for each route. The starting- and subsequent concentrations were taken from the series: 100% (undiluted), 50%, 20%, 10%, 5%,2%,1% and if needed, further lower concentrations using the same steps. The test system and procedures were identical to those used during the main study, unless otherwise specified. The five animals selected were between 4 and 9 weeks old and the body weights of some animals did exceed 500 grams. No body weights were determined at termination. .

Intradermal injections:
Initially, a series of four test substance concentrations was used; the highest concentration being the maximum concentration that could technically be injected. Each of two animals received two different concentrations in duplicate (0.1 ml/site) in the clipped scapular region. The resulting dermal reactions were assessed 24 and 48 hours after treatment. Based on the results in the initially treated animals, one additional animal was treated in a similar manner with two lower concentrations at a later stage.

Epidermal application:
A series of four test substance concentrations was used: the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5 ml each) per animal to the clipped flank, using Metallline patches# (2x3 cm) mounted on Medical tape, which were held in place with Micropore tape* and subsequently Caban elastic bandage*. The initially used animals receiving intradermal injections were treated with the lowest concentrations and two further animals' with the highest concentrations. After 24 hours, the dressing was removed and the skin cleaned of residual test substance. The resulting dermal reactions were assessed for irritation 24 and 48 hours after exposure.
* Suppliers: Lohmann GmbH, Neuwied, Germany (Coban) and 3M, St. Paul, Minnesota, U.S.A. (Metalline and Mlcropore)

MAIN STUDY

INDUCTION - Experimental animals

Day 1 The scapular region was clipped and three pairs of intradermal injections (0.1 ml/site) were made In this area as follows:
A) A 1:1 W/W mixture of Freunds' Complete Adjuvant (Difco, Detroit, U.S.A.) with water for injection (Fresenius AG, Bad Homburg, Germany).
B) The test substance at a 5% concentration.
C) A 1:1 wlw mixture of the test substance, at twice the concentration used in (6) and Freunds' Complete Adjuvant.
Note: One of each pair was on each side of the midline and from cranial A) to caudal C).

Day 3. The dermal reactions caused by the intradermal injections were assessed for irritation.

Day 7. The scapular area between the injection sites was clipped and subsequently rubbed with 10% sodium-dodecyl-sulfate (SOS, Boom, Meppel, The Netherlands) in Vaseline using a spatula. This concentration of SDS provokes a mild inflammatory reaction.

Day 8: The 10% SDS treated area between the injection sites was treated with 0.5 ml of a 100% test substance concentration using a Metalline patch (2x3 em) mounted on Medical tape, which was held in place with Micropore tape and subsequently Coban elastic bandage. The dressing was removed after 48 hours exposure, the skin cleaned of residual test substance and the dermal reactions caused by the epidermal exposure were assessed for irritation.

INDUCTION - Control animals
The control animals were treated as described for the experimental animals except that, instead of the test substance, vehicle alone was administered.

CHALLENGE - All animals

Day 22: One flank of all animals was clipped and treated by epidermal application of a 100% test substance concentration and the vehicle (0.15 ml each), using Patch Test Plasters (Leukotest ®, Beiersdorf Medical, Almere, The Netherlands). The patches were held in place with Micropore tape and subsequently Caban elastic bandage. The dressing was removed after 24 hours exposure and the skin cleaned of residual test substance and vehicle. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing.
Challenge controls:
yes
Positive control substance(s):
no

Results and discussion

Positive control results:
RESULTS

PRELIMINARY IRRITATION STUDY
Preliminary tests on intradermal injections and epidermal exposures were conducted for the selection of suitable test substance concentrations for the main study. Based on the results, the test substance concentrations selected for the Main Study were a 5% concentration for the intradermal induction and a 100% concentration for the epidermal induction exposure.

No signs of irritation were observed to the highest test substance concentration epidermally tested. Therefore, the test site of all animals was treated with 10% SDS approximately 24 hours before the epidermal induction in the main study, to provoke a mild inflammalory reaction. A 100% test substance concentration was selecled for the challenge phase.

MAIN STUDY

Induction phase
The skin effects caused by the intradermal injections and epidermal exposure during the induction phase recorded. The reactions noted in the experimental animals after the epidermal induction exposure were considered to be enhanced by the SDS treatment.

Challenge phase
Skin reactions of grade 4 were observed in all experimental animals in response to the 100% test substance concentration. No skin reactions were evident in the control animals. Eschar formation or scabs were seen in all treated skin sites among the experimental animals.

Toxicity I Mortality
One experimental animal was removed from the sludy on day 22 following the observation of piloerection, emaciation, watery discharge from the eyes, rales, dirty teeth and the animal felt cold. Macroscopic post-mortem examination showed scabs and/or alopecia on back and abdomen and pelvic dilation of the left kidney. It was considered that the death of this animal was incidental and that the study outcome, based on the healthy surviving animals, was not adversely affected. No further mortality occurred and no further symptoms of systemic toxicity were observed in the remaining animals of the main study.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
Normal
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: Normal.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
0.15 ml of 100% of the test substance
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
Eschar formation and scabs, Grade 4 (max)
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 0.15 ml of 100% of the test substance. No with. + reactions: 9.0. Total no. in groups: 10.0. Clinical observations: Eschar formation and scabs, Grade 4 (max).
Reading:
2nd reading
Hours after challenge:
600
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
Normal
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 600.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: Normal.
Reading:
2nd reading
Hours after challenge:
600
Group:
test group
Dose level:
.15 ml of 100% of the test substance
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
Eschar and scab formation, Grade 4 (max)
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 600.0. Group: test group. Dose level: .15 ml of 100% of the test substance. No with. + reactions: 9.0. Total no. in groups: 10.0. Clinical observations: Eschar and scab formation, Grade 4 (max).

Any other information on results incl. tables

RESULTS

 

PRELIMINARY IRRITATION STUDY

Preliminary tests on intradermal injections and epidermal exposures were conducted for the selection of suitable test substance concentrations for the main study. Based on the results, the test substance concentrations selected for the Main Study were a 5% concentration for the intradermal induction and a 100% concentration for the epidermal induction exposure.

 

No signs of irritation were observed to the highest test substance concentration epidermally tested. Therefore, the test site of all animals wastreatedwith 10% SDS approximately 24 hours before the epidermal induction in the main study, to provoke a mildinflammaloryreaction. A 100% test substance concentration was selecled for the challenge phase.

 

MAIN STUDY

 

Induction phase

The skin effects caused by the intradermal injections and epidermal exposure during the induction phase recorded. The reactions noted in the experimental animals after the epidermal induction exposure were considered to be enhanced by the SDS treatment.

 

Challenge phase

Skin reactions of grade 4 were observed in all experimental animals in response to the 100% test substance concentration. No skin reactions were evident in the control animals. Eschar formation or scabs were seen in all treated skin sites among the experimental animals.

 

ToxicityIMortality

One experimental animal was removed from thesludy on day 22 following the observation of piloerection, emaciation, watery discharge from the eyes, rales, dirty teeth and the animal felt cold. Macroscopic post-mortem examination showed scabs and/or alopecia on back and abdomen and pelvic dilation of the left kidney. It was considered that the death of this animal was incidental and that the study outcome, based on the healthy surviving animals, was not adversely affected. No further mortality occurred and no further symptoms of systemic toxicity were observed in the remaining animals of the main study.

 

Body Weights

Body weights and body weight gain of experimental animals remained in the same range as controlsover the study period.

 

Grading Irritation Reactions:

Erythema and eschar formation:

No erythema .......................................................................................................................... 0

Slightly erythema (barely perceptible) ................................................................................ I

Well-defined .erythema .......................................................................................................... 2

Moderate erythema................................................................................................................ 3

Severe erythema (beet redness) to slight eschar formation (injuries in depth)........ 4

 

Oedema fonnatlon:

No oedama .............................................................................................................................0

Slighl oedema (barely perceptible) .................................................................................... 1

Well-defined oedema (edges: of area well-definedby definite raising)....................... 2

Moderate oedema (raised approximately1 millimeter) .................................................. 3

Severe oedema (raised more than 1 millimeter and extending beyond the area of exposure)...... 4

Intradermal reactions were assessed for erythema only or, if necrosis is present, the diameter of necrosis.

 

Grading Challenge Reactions:

No visible change.............................................................. ................................................... 0

Discrete or patchy erythema................................................................................................ 1

Moderate and confluent erythema...................................................................................... 2

Moderate erythema and swellIng................................................................................ , .... 3

Intense erythema and swelling........................................................................................... 4

After the end of the study all animals were killed by asphyxiation using an oxygen/carbon dioxide procedure.

 

INTERPRETATION

 

The results for the experimental animals at the challenge phase were compared with the results for the control animals. Positive skin reactions (grade 1 or more) were considered signs of sensitlsation, provided that such reactions were not observed or were less persistent in the control group. A sensitisation rate (%) was calculated as follows: the number of sensitised animals as a proportion of the total number of animals in the experimental group. The results were evaluated according to the EC criteria for classification and labeling requirements for dangerous substances and preparations (Guidelines in Commission Directive93/21/EEC).

CONCLUSION

The skin reaction observed in response to a 100% test substance concentration in all (of the 9) experimental animals in the challenge phasewere considered indicative of sensitisation, based on the absence of any response in the control animals. These results indicate a sensitisation rate of 100 per cent.

 

Based on these results and according to the EC criteria for classification and labeling requirements for dangerous substances and preparations (Guidelines in Commission Directive93f211EEC),TRIGONOX 448 should be labelled as: may cause sensitisation by skin contact (R43).

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The skin reactions observed in response to a 100% test substance concentration in all (of the nine) experimental animals in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals. These results indicate a sensitisation rate of 100 per cent.
 
Based on these results and according to the EC criteria for classification and labeling requirements for dangerous substances and preparations (Guidelines in·Commission Directive93/21/EEC),TRIGONOX 446 should be labelled as: may cause sensitisation by skin contact (R43). 
Executive summary:

SUMMARY: Assessment for contact Hypersensitivity to trigonox 44B in the Albino Guinea Pig (Maximization Test).

 

The study was carried out based on the guidelines described in: EC Commission Directive 96154/EC,Part B.6, 'Skin Sensitisation" and OECD No. 406, "Skin SensHisation', and based on the method described by Magnusson and Kligman, "Allergic Contact Dermatitis in the Guinea Pig - Identification of Contact Allergens '.

 

Test substance concentrations selected for the main study were based on the results of a preliminary study.

 

In the main study, ten experimental animals were intradermally injected with a 5% concentration and epidermally exposed to a 100% concentration. Five control animals were similarly treated, but with vehicle alone (water). Approximately 24 hours before the epidermal induction exposure all animals were treated with 1 0% SDS. Two weeks after the epidermal application the animals were challenged with a 100% test substance concentration or the vehicle.

 

In the challenge phase, skin reactions of grade 4 were observed in all (01 nine) experimental animals in response to the 100% test substance concentration. No skin reactions were evident in the control animals. Eschar formation or scabs were seen in all treated skin sites among the experimental animals. One experimental animal was removed from the study on day 22 following the observation of piloerection, emaciation, watery discharge from the eyes, rales, dirty teeth and the animal felt cold. Macroscopic post-mortem examination showed scabs and/or alopecia on back and abdomen and pelvic dilation of the left kidney.

It was considered that the death of this animal was incidental and that the study outcome, based on the healthy surviving animals, was not adversely affected. No further mortality occurred and no further symptoms of systemic toxicity were observed in the remaining animals of the main study. The skin reactions observed in response to a 100% test substance concentration in all (of the nine) experimental animals in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals. These results indicate a sensitisation rate of 100 per cent.

 

Based on these results and according to the EC criteria for classification and labeling requirements for dangerous substances and preparations (Guidelines in·Commission Directive93/21/EEC),TRIGONOX 446 should be labelled as: may cause sensitisation by skin contact (R43).