2,6,6-trimethylcyclohex-2-ene-1,4-dione

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to OECD 406 and EU Directive 92/69/EC. Study conducted under GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Test material

Test material information

Constituent 1

Reference substance name:

2,6,6-trimethylcyclohex-2-ene-1,4-dione

EC Number:

214-406-2

EC Name:

2,6,6-trimethylcyclohex-2-ene-1,4-dione

Cas Number:

1125-21-9

Molecular formula:

C9H12O2

IUPAC Name:

2,6,6-trimethylcyclohex-2-ene-1,4-dione

Constituent 2

Reference substance name:

oxoisophoron

IUPAC Name:

oxoisophoron

Constituent 3

Reference substance name:

2,6,6-Trimethyl-2-cyclohexen-1,4-dion

IUPAC Name:

2,6,6-Trimethyl-2-cyclohexen-1,4-dion

Details on test material:

- name of test substance: Oxoisophoron
- chemical name: 2,6,6-Trimethyl-2-cyclohexene-1,4-dion
- substance number: 96/87
- CAS number: 1125-21-9
- degree of purity/content: 98.87%
- date of manufacturing: 18-may-1995
- physical state/appearance: solid, yellow (at room temperature); liquid, yellowish (at 50 °C)
- storage conditions: room temperature

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Pirbright white

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River GmbH, Wiga, Kisslegg, FRG
- Age at study initiation: young adult animals
- Weight at study initiation: 339 - 400 g
- Housing: in group; Makrolon, type IV cage
- Diet (e.g. ad libitum): Kliba Labordiaet 341 (Kaninchen-Meerschweinchen-Haltungsdiaet) ad libitum
- Water (e.g. ad libitum): ad libitum; tap water; about 2 g of ascorbic acid per 10 L water was added to the drinking water twice aday.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 h/12 h

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

other: intradermal and percutaneous

Vehicle:

olive oil

Induction 2

Concentration / amount:

Intradermal induction:
Test substance 5% in olive oil DAB 10 or in Freund's adjuvant/0.9% aqueous NaCl-solution (1:1) or olive oil DAB 10

Percutaneous induction:
Test substnace 50% in olive oil DAB 10 or olive oil DAB 10

Challenge:
Test substance 25% in olive oil DAB 10 or olive oil DAb 10

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

olive oil

Challenge 2

Concentration / amount:

Intradermal induction:
Test substance 5% in olive oil DAB 10 or in Freund's adjuvant/0.9% aqueous NaCl-solution (1:1) or olive oil DAB 10

Percutaneous induction:
Test substnace 50% in olive oil DAB 10 or olive oil DAB 10

Challenge:
Test substance 25% in olive oil DAB 10 or olive oil DAb 10

No. of animals per dose:

control group: 5
test group: 10

Details on study design:

1) Main test
Number of animals per control group: 5
Number of animals of the test group: 10


2) Induction
Intradermal induction:
- 6 intradermal injections in groups of twa per animal

Injections for the test group:
A) front row: 2 injections each of 0.1 ml Freund's adjuvant without test substance emulsified with 0.9% aqueous NaCl-solution in a ratio of 1 :1
B) middle row: 2 injections each of 0.1 ml of the test substance formulation
C) back row: 2 injections each of 0.1 ml Freund's adjuvant / 0.9% aqueous NaCl-solution (1 : 1) with test substance

Injections for control groups 1 and 2:
- The animals were given the same injections (A, B, C) but without test substance, only with the formulating agent.

Site of application:
- shoulder

Readings:
- 24 h after the beginning of application

Assessment of the skin findings:
- analogous to the pretest

Percutaneous induction:
- Percutaneous induction was carried out one week after intradermal induction.

Amount applied:
2 x 4 cm filter paper strips containing the test substance formulation were applied to the skin of the shoulder under an occlusive ciressing. The filter paper strip was soaked in the test substance formulation. The dressing consisted of rubberized linen patches (4 x 6 cm from Russka) and Fixomull® Stretch (adhesive fleece) from Beiersdorf AG. The control groups were treated analogously to the test group but only with the solvent without the test substance.

Duration of exposure:
- 48 hours

Site of application:
- shoulder, same area as in the case of the previous intradermal application

Readings:
- 48 h after the beginning of application

Assessment of skin findings:
-analogous to the pretest


3) Challenge
The challenge was performed 14 days after the percutaneous induction.

Amount applied:
2 x 2 cm filter paper strips containing the test substance formulation were applied to the skin of the flank under an occlusive dressing. The filter paper strip was soaked in the test substance formulation. The dressing consisted of rubberized linen patches (4 x 4 cm from Russka), patches of Idealbinde (5 x 5 cm from Pfälzische Verbandstoff-Fabrik) and Fixomull®D Stretch (adhesive fleece) from Beiersdorf AG.

-The test group and control group 1 were treated with the test substance formulation (control group 2 remained untreated).

Duration of exposure:
- 24 hours

Site of application:
- intact flank

Readings:
- 24 and 48 h after the removal of the patch

Assessment of skin findings:
- analogous to the pretest


4) Positive control

A positive control (reliability check) with a known sensitizer is not included in this study. However, a separate study is performed twice a year in the laboratory.
The positive control with Alpha-Hexylcinnamaldehyde techn. 85% showed that the test system was able to detect sensitizing compounds under the laboratory
conditions chosen.

Positive control substance(s):

yes

Remarks:

alpha-hexylcinnamaldehyde

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25%

No. with + reactions:

6

Total no. in group:

10

Clinical observations:

Well-defined erythema in 3/10 (1 of these animals additionally with very slight edema); very slight erythema in 3/10.

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25%. No with. + reactions: 6.0. Total no. in groups: 10.0. Clinical observations: Well-defined erythema in 3/10 (1 of these animals additionally with very slight edema); very slight erythema in 3/10..

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25%

No. with + reactions:

5

Total no. in group:

10

Clinical observations:

Very slight erythema in 4/10; well-defined erythema in 1/10.

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25%. No with. + reactions: 5.0. Total no. in groups: 10.0. Clinical observations: Very slight erythema in 4/10; well-defined erythema in 1/10..

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

vehicle (olive oil DAB 10)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

none

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: vehicle (olive oil DAB 10). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: none.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

vehicle (olive oil DAB 10)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

none

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: vehicle (olive oil DAB 10). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: none.

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

none

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. Clinical observations: none.

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

none

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: 5.0. Clinical observations: none.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

Based on the results of this study, the test substance 2,6,6-trimethylcyclohex-2-ene-1,4-dione / oxoisophoron has to be classified skin sens. Cat. 1 and R43 according to Regulation (EC) No. 1272/2008 and EU Directive 67/548/EEC, respectively.