6'-(dibutylamino)-3'-methyl-2'-(phenylamino)spiro[isobenzofuran-1(3H),9-(9H)-xanthen]-3-one

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 February to 12 March 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

not specified

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

An adequate and relevent in vivo study is available.

Test material

Test material information

Constituent 1

Reference substance name:

ODB-2

IUPAC Name:

ODB-2

Constituent 2

Reference substance name:

6'-(dibutylamino)-3'-methyl-2'-(phenylamino)spiro[isobenzofuran-1(3H),9-(9H)-xanthen]-3-one

EC Number:

403-830-5

EC Name:

6'-(dibutylamino)-3'-methyl-2'-(phenylamino)spiro[isobenzofuran-1(3H),9-(9H)-xanthen]-3-one

Cas Number:

89331-94-2

Molecular formula:

C35 H36 N2 O3

IUPAC Name:

6'-(dibutylamino)-3'-methyl-2'-(phenylamino)-3H-spiro[2-benzofuran-1,9'-xanthen]-3-one

Details on test material:

- Name of test material (as cited in study report): ODB-2
- Substance type: Colour precursor for heat sensitive record sheets
- Physical state: White powder
- Analytical purity: > 99%
- Impurities (identity and concentrations): No data
- Purity test date: No data
- Lot/batch No.: 61-24101
- Expiration date of the lot/batch: No data
- Stability under test conditions:
- Storage condition of test material: Ambient in the dark
- Other: date received 26 January 1988

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Commercial laboratory animal supplier
- Age at study initiation:
- Weight at study initiation: 460 - 536 g
- Housing: suspended cages with wire mesh floors
- Diet: Vitamin C-enriched guinea-pig diet F.D.1.,ad libitum. Hay was given weekly.
- Water: ad libitum
- Acclimation period: 19 days

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

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

other: Alembicol D

Induction 2

Concentration / amount:

Induction
Intradermal injection: 0.05% w/w in Alembicol D.
Topical application: 50% w/w in Alembicol D.

Challenge
Topical application: 50% and 25% w/w in Alembicol D.

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: Alembicol D

Challenge 2

Concentration / amount:

Induction
Intradermal injection: 0.05% w/w in Alembicol D.
Topical application: 50% w/w in Alembicol D.

Challenge
Topical application: 50% and 25% w/w in Alembicol D.

No. of animals per dose:

20

Details on study design:

RANGE FINDING TESTS:
The intradermal and topical irritancy of a range of dilutions of ODB-2 was investigated to identify (a) irritant test substance concentrations suitable for the induction phase of the main study and (b) non-irritant concentrations by the topical route of administration for the challenge phase.

A concentration of 50% in AlembicolD was the maximum practical concentration that could be prepared and dosed topically. Based on the results of the preliminary investigations, the following concentrations of ODB-2 were selected:

Induction
Intradermal injection: 0.05% w/w in Alernbicol D.
Topical application: 50% w/w in Alernbicol D.

Challenge
Topical application: 50% and 25% w/w in Alernbicol D.

Alernbicol D is a product of coconut oil. By fractionating the fatty acids of coconut oil and re-esterifyinq with glycerine, a Medium Chain Triglyceride (MCT) Oil is produced. Alernbicol D is such an oil. It is advantageous in this type of test in that it is stable, has a low viscosity and surface tension and is easily absorbed into the skin. These properties made it ideal as a vehicle for intradermal injections and topical applications.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 1 week
- Test groups: 1
- Control group: yes, 10 animals
- Site: right and left dorsal
- Frequency of applications:
- Duration: two weeks
- Concentrations:
1. Freund's complete adjuvant* was diluted with an equal volume of water for irrigation.
2. ODB-2, 0.05% w/w in Alembicol D.
3. ODB-2, 0.05% w/w in a 50 : 50 mixture of Freund's complete adjuvant and Alembicol D.

B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge:
- Exposure period: 3 days
- Test groups: 20 animals
- Control group: 10 animals
- Site: Shaved flanks.
- Concentrations: 0.2 ml of ODB-2, 50% w/w in Alembico1 D and applied to an anterior site on the flank. ODB-2, 25% w/w in Alembicol D was applied in a similar manner to a posterior site.
- Evaluation (hr after challenge): 24, 48 and 72 hours

OTHER:

Challenge controls:

The test and control animals were challenged topically two weeks after the induction period using ODB-2, 50% and 25% w/w in Alembicol D.

Positive control substance(s):

yes

Remarks:

The sensitivity of the guinea-pig strain used is checked periodically at H.R.C. with formalin, a known sensitiser.

Results and discussion

Positive control results:

1988; 10 animals tested all positive with formalin.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

72

Group:

negative control

Dose level:

ODB-2, 25% and 50%w/w in Alembicol D

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 72.0. Group: negative control. Dose level: ODB-2, 25% and 50%w/w in Alembicol D . No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 2

Key result

Reading:

1st reading

Hours after challenge:

72

Group:

test chemical

Dose level:

ODB-2, 25% and 50% in Alembicol D.

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 72.0. Group: test group. Dose level: ODB-2, 25% and 50% in Alembicol D.. No with. + reactions: 0.0. Total no. in groups: 20.0.

Results 3

Reading:

1st reading

Hours after challenge:

72

Group:

positive control

Dose level:

10% formaldehyde aqueous solution (Formalin)

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

not reported

Remarks on result:

positive indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In this screening test, performed in twenty albino guinea-pigs, ODB-2 did not produce evidence of delayed contact hypersensitivity.