4-(4-nitrophenylazo)-2,6-di-sec-butyl-phenol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This study was carried out in accordance with the OECD Guideline No. 406, "Skin Sensitisation", the EEC Directive B4/449/EEC, Part B.6, "Skin Sensitisation" and in accordance with the method described by Magnusson and Kligman, "Allergic Contact Dermatitis in the Guinea Pig - Identification of Contact Allergens" and in accordance with the Principles of Good Laboratory Practices (GLP).

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

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

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Study conducted prior to "LLNA" requirement.

Test material

Test material information

Constituent 1

Reference substance name:

4-(4-nitrophenylazo)-2,6-di-sec-butyl-phenol

EC Number:

410-610-2

EC Name:

4-(4-nitrophenylazo)-2,6-di-sec-butyl-phenol

Cas Number:

111850-24-9

Molecular formula:

C20 H25 N3 O3

IUPAC Name:

2,6-bis(butan-2-yl)-4-[2-(4-nitrophenyl)diazen-1-yl]phenol

Test material form:

other: dark red-brown liquid

Details on test material:

- Name of test material (as cited in study report): Mortrace SB Conc.
- Physical state: dark red-brown liquid
- Analytical purity: concentrate
- Lot/batch No.: MR 26592 SBC
- Expiry date: June 03, 1993
- Stability under storage conditions: stable
- Stability in vehicle: stability in corn oil not indicated by the sponsor
- Storage condition of test material: at room temperature protected from light

In vivo test system

Test animals

Species:

guinea pig

Strain:

Himalayan

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: BRL Ltd., Basel, Switzerland
- Age at study initiation: approximately 10 weeks
- Weight at study initiation: 435 - 525 grams
- Housing: grouo housed of 2 animals/cage
- Diet: ad libitum access to standard guinea pig diet, including ascorbic acid. In addition hay was provided once a week
- Water: ad libitum
- Acclimation period: at least 5 days before start of the treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Humidity (%): 55%
- Air changes (per hr): 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

corn oil

Induction 2

Concentration / amount:

Preliminary study - 0.05 ml of 50%, 25%, 10% and 5% (w/w) concentration of the test substance in corn oil
Main study - 0.05 ml of 25%, 10% and 5% (w/w) concentration of the test substance in corn oil

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

corn oil

Challenge 2

Concentration / amount:

Preliminary study - 0.05 ml of 50%, 25%, 10% and 5% (w/w) concentration of the test substance in corn oil
Main study - 0.05 ml of 25%, 10% and 5% (w/w) concentration of the test substance in corn oil

No. of animals per dose:

Preliminary study - 4 females
Main study -
Experimental group - 20 females
Control group - 10 females

Details on study design:

Preliminary study -
Intradermal injections:
Four intradermal injections (0.1 m1/site) were made into the clipped shoulder region of one guinea pig with a S% (w/w) concentration of the test substance
in corn oil. The resulting dermal reactions were assessed 24 and 48 hours later. The following parameters were recorded:
Erythema: scored according to the scale described below,
Necrosis: if present yes or no,
Diameter: (mm) of effect (erythema, necrosis or discolouration).

Epidermal applications:
The intradermally injected animal was also treated epidermally at the shaved left flank with 0.S ml of a 50% (w/w) concentration of the test substance in corn oil using a Scotchpak~non-woven patch (2.S x 2.2 cm) mounted on Micropore tape (both from 3M, st. Paul, U.S.A.) and held in place with Coban elastic
bandage (3M, St. Paul, U.S.A.). After 24 hours, the dressings and residual test substance were removed using a moistened tissue. The treated skin was assessed for erythema and oedema 24 and 48 hours after bandage removal on a numerical basis according to the Draize scale.
Three other animals were shaved on the left flank and exposed to 0.05 ml of a 50%, 2S%, 10% and S% (w/w) concentration of the test substance in corn oil, occlusively administered by means of Square chambers (v.d. Bend, Brielle, The Netherlands) mounted on Micropore tape and fixed in place by means of Coban
elastic bandage. This procedure ensured the intensive contact of the test substance. After 24 hours, the dressings and residual test substance were removed using a moistened tissue.
The reaction sites were assessed for erythema and oedema on a numerical basis according to the Draize scale, 24 and 48 hours after bandage removal. Immediately after the 24 hour skin reading the treated areas were shaved.

Main study -
Induction -
Intradermal injections -
On day 1 an area of the dorsal skin from the scapular region (approximately 4 x 6 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 ml/site) were made at the border of a 2 x 4 cm area in the clipped region as follows:
A) The test substance dissolved to 5% (w/w) with corn oil.
B) Freunds' Complete Adjuvant (FCA, Difco, Detroit, U.S.A.), 50:50 with distilled water for injection (pyrogen free, Ferensius AG, Bad Homburg, Germany).C) The test substance, at twice the concentration used in (A), emulsified in a 50:50 mixture of Freunds' Complete Adjuvant.
Epidermal applications -
Seven days after the intradermal injections, the scapular area (approximately 6 x 8 cm) was clipped and shaved free of hair. A Scotchpak-non-woven patch (2 x 4 cm) mounted on Micropore tape was applied with 0.5 ml of a 50% (w/w) concentration of the test substance in corn oil and placed between the injection sites of the test animals. The Micropore tape was firmly secured, wrapped around the trunk uf the animal and secured with Coban elastic bandage. After 48 hours, the dressings and residual test substance were removed using a moistened tissue.
The epidermal application procedure ensured intensive contact of the test substance.
The guinea pigs of the control group were treated as described above by the intradermal and epidermal inductions with the omission of test substance. Reaction sites were assessed for erythema and oedema immediately after removal of the dressings.
Challenge -
The test and control guinea pigs were challenged two weeks after the epidermal induction application.
Hair was clipped and shaved from a 5 x 5 cm area on the left flank of each guinea pig. A volume of 0.05 m1 of each of the following three test substance concentrations and the vehicle were applied using Square chambers attached to Micropore tape:
a = 25% (w/w) in corn oil.
b = 10% (w/w) in corn oil.
c = 5% (w/w) in corn oil.
d = corn oil.
The patches were placed on the shaved area, the Micropore tape firmly secured around the trunk of the animals and held in place by Coban elastic bandage. The dressings and residual test sUbstance were removed after approximately 24 hours, using a moistened tissue. The sites were assessed for redness and swelling 24 and 48 hours after removal of the dressings, using the numerical grading system described below (modified from Kligman A.M., J. Invest. Dermatol. 47, 1966). The test sites were re-shaved with an electric razor after the first reading. All animals were sacrificed at the end of the test period by intraperitoneal
injection of Euthesate (A.U.V., Boxmeer, The Netherlands), approximately 2ml/animal.

Challenge controls:

as described above

Positive control substance(s):

yes

Remarks:

formaldehyde

Results and discussion

Positive control results:

Positive results were observed in the experimental animals after the challenge with 0.2% (w/w) formaldehyde in distilled water.

In vivo (non-LLNA)

Results

Results 1

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25%

No. with + reactions:

17

Total no. in group:

20

Clinical observations:

Reactions were characterised by red spots to eschar/crust formation, swelling and scaliness. All challenge skin sites showed yellow disciolouration caused by the test sUbstance which made skin reading difficult

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25%. No with. + reactions: 17.0. Total no. in groups: 20.0. Clinical observations: Reactions were characterised by red spots to eschar/crust formation, swelling and scaliness. All challenge skin sites showed yellow disciolouration caused by the test sUbstance which made skin reading difficult.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10%

No. with + reactions:

9

Total no. in group:

20

Clinical observations:

same as above

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10%. No with. + reactions: 9.0. Total no. in groups: 20.0. Clinical observations: same as above.

Results 3

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

5%

No. with + reactions:

9

Total no. in group:

20

Clinical observations:

same as above

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 5%. No with. + reactions: 9.0. Total no. in groups: 20.0. Clinical observations: same as above.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

see above

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: see above.

Results 5

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

0 %

No. with + reactions:

0

Total no. in group:

20

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

0.1%

No. with + reactions:

11

Total no. in group:

20

Results 7

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

0.2%

No. with + reactions:

19

Total no. in group:

20

Any other information on results incl. tables

None

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Taking into account the intensity of the responses and comparing these with the skin reactions seen in the control animals, seventeen animals showed a positive reaction in response to the 25% concentration, nine animals in response to the 10% concentration and nine animals in response to the 5% concentration.
These results lead to a sensitisation rate of 85 per cent, which indicated that MORTRACE S8 CONC. had extreme sensitizing properties in this test applying the rating of allergenicity described by Kligman A.M. (1966).

Executive summary:

The purpose of the study was to obtain information on the potential of MORTRACE SB CONC. to induce delayed contact hypersensitivity (skin sensitization) in the guinea pig after intradermal and epidermal exposures. This study was carried out in accordance with the OECD Guideline No. 406, "Skin Sensitisation", the EEC Directive B4/449/EEC, Part B.6, "Skin Sensitisation" and in accordance with the method described by Magnusson and Kligman, "Allergic Contact Dermatitis in the Guinea Pig - Identification of Contact Allergens". After identification of the slightly irritating and the non-irritating test substance concentrations in the preliminary study, a main study was performed with the selected test substance concentrations. The experimental animals were intradermally injected with a 5% concentration and epidermal exposed to a 50% concentration, while the control animals were similarly treated, but with the vehicle only. Immediately after the epidermal exposure, the skin irritation was scored. Two weeks after the epidermal application all animals were challenged with test substance concentrations of 25%, 10% and 5%, and the vehicle (corn oil). The challenge reactions were assessed 24 and 48 hours after bandage removal. The epidermal exposure of MORTRACE SB CONC. in the induction phase resulted in no marked skin irritation. The treated skin area was yellow discolored by the test substance. The epidermal exposure of MORTRACE SB CDNC. in the challenge phase resulted in seventeen positive sensitization reactions in response to the 25% test substance concentration.

Under the conditions used in this study, MORTRACE SB CONC. resulted in a sensitization rate of 85 per cent. Applying the rating of a1lergenicity described by Kligman A.M. (1966) on the results obtained in this test, MORTRACE SB CONC. is considered to have extreme sensitizing properties. Based on these results and according to the EEC criteria for classification and labeling requirements for dangerous substances and preparations (EEC Directive 91/325/EEC, Amendment to Annex VI of the EEC Council Directive 67/548/EEC), MORTRACE SB CONC. should be labeled as a skin sensitizer.