8-[4-(3,4-Difluorophenyl)-3-cyclohexene-1-yl)-1,4-dioxaspiro[4.5]decane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 13, 2002 - September 12, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was performed in compliance with the Good Laboratory Practice (GLP) regulations (revised in 1997, ENV/MC/CHEM(98)17). The method followed that described in the OECD Guidelines for Testing of Chemicals, Updated Guideline No 406 Skin Sensitisation (adopted 17 Julyl 1992).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted on July 1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

Commission Directive 96/54/EEC of 30 July 1996 adapting to technical progress for the 22nd time Council Directive 67/548/EEC on the approximation of laws, regulations and administrative provisions relating to the classification, packing and labeling of dangerous substances
(Official Journal No. L 248 A, September 30, 1996)
Part B6. " Skin sensitization"

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The data have been generated before the OECD 429 protocol was mandatory for REACH.

Test material

Test material information

Constituent 1

Reference substance name:

8-[4-(3,4-difluorophenyl)cyclohex-3-en-1-yl]-1,4-dioxaspiro[4.5]decane

EC Number:

604-591-1

Cas Number:

147622-83-1

Molecular formula:

C20H24F2O2

IUPAC Name:

8-[4-(3,4-difluorophenyl)cyclohex-3-en-1-yl]-1,4-dioxaspiro[4.5]decane

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test material was prepared using liquid paraffin or polyethylene glycol 400 or an FCA preparation (Freund's complete adjuvant) directly prior to administration.

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: HsdPoc:DH

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen
- Females (if applicable) nulliparous and non-pregnant:yes
- Microbiological status of animals, when known: no data
- Age at study initiation: about 5 weeks
- Weight at study initiation: Mean body weight 339g (vehicle group) and 349g (Test material group)
- Housing: Five guinea pigs were housed in type GM/5 (EBECO) Makrolon cages with a shelter and placed on mobile racks. The animals were kept on conventional softwood granulate as bedding. The bedding was changed twice per week. The softwood granulate was analytically checked by independent laboratories.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 7 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 22 °C
- Humidity (%): 50 to 79 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hour light – 12 hour dark regime

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

paraffin oil

Induction 2

Concentration / amount:

see details on study design

Challenge

Challenge 1

Route:

epicutaneous, semiocclusive

Vehicle:

polyethylene glycol

Challenge 2

Concentration / amount:

see details on study design

No. of animals per dose:

Total: 20 females Pre-test: 5 females Control group: 5 females Test group 10 females

Details on study design:

RANGE FINDING TESTS:

To determine the concentrations suitable for the main study, a pretest with single intradermal or topical administrations of the vehicle and ofthe test item preparations was performed. Intradermal (i.d.) injections were given to one animal. Four further animals were used to determine the topical concentrations for the main study. Two of the guinea pigs were exposed for 48 hours without pretreatment to determine the concentration for the dermal induction. The other two animals were treated according to the control group including intradermal injections with FCA. One week after topical induction the two animals of the pretest group were exposed for 24 hours to different test material concentrations to find out the challenge concentration. In the pretest the following concentrations were used:

Test item with liquid paraffin as vehicle:
without pretreatment: intradermal injection: 10, 5, and 1 g/L

Test item with polyethylene glycol 400 as vehicle:
without pretreatment: topical exposure (48 hours): 400, 200, 100 and 10 g/L
pretreatment with FCA: topical exposure (24 hours): 200, 100, 50 and 10 g/L


MAIN STUDY
A. INDUCTION STAGE 1 (intradermal injection)

After shaving of the shoulder region, six intradermal injections were given (three on each side of the spinal column in a total area of 2 x 4 cm). The guinea pigs received the following injections:

Group 1 (negative control group)
cranial: 0.1 ml Freund's complete adjuvant + sodium chloride solution
medial: 0.1 ml Liquid paraffin
caudal: 0.1 ml Freund's complete adjuvant + sodium chloride solution

Group 2 (test material group)
cranial: 0.1 ml Freund's complete adjuvant + sodium chloride solution
medial: 0.1 ml Test item (1 g/L ad liquid paraffin)
caudal: 0.1 ml Freund's complete adjuvant with test item
+ sodium chloride solution
(1.0g test material/L preparation)

A. INDUCTION STAGE 2 (topical application)

One week after the intradermal injections, the shoulder area of the guinea pigs was shaven again and covered with a filter paper patch of about 8 cm² fully soaked with
1 ml of the test material preparation or the vehicle. The patches were attached for 48 hours under occlusive conditions.

Group 1: Polyethylene glycol 400
Group 2: Test item (200 g/L ad polyethylene glycol 400)


B. CHALLENGE EXPOSURE
Two weeks after the topical induction the challenge was performed by fixing filter papers of about 4 cm2 fully loaded with 0.5 ml of thetest item preparation or soaked with 0.5 ml polyethylene glycol 400 to the shaven side of the animals.

Group 1: Polyethylene glycol 400 (undiluted)

Group 2: Art. 272318 (100 g/L polyethylene glycol 400)

The patches were fixed for 24 hours under occlusive conditions by a self-adhesive fabric.

Positive control substance(s):

yes

Remarks:

Hexylcinnamaldehyde

Results and discussion

Positive control results:

Under the given experimental conditions and with respect to the evaluation criteria, the test material alpha-Hexylcinnamaldehyde exhibits a skin-sensitization potential.

Positive reactions were observed in 30% or 50 % of the animals. In summary, under the conditions of the maximization test and with respect to the evaluation criteria, the test material exhibits a skin-sensitization potential. This confirms the sensitivity and reliability of the GPMT performed at the Institute of Toxicology, Merck KGaA, with the guinea pig strain: Hsd Poc:DH. (Heusener, 2002)

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

1 g/L (intradermal induction I), 200 g/L (topical induction II), 100 g/L (topical challenge)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no signs of toxicity

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

1 g/L (intradermal induction I), 200 g/L (topical induction II), 100 g/L (topical challenge)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no signs of toxicity

Remarks on result:

no indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the given experimental conditions, the test material induced no reactions.

Executive summary:

Conclusion

Under the given experimental conditions, the test material induced no reactions.