5(or 6)-methyl-7(or 8)-(1-methylethyl)bicyclo[2.2.2]oct-5-ene-2-carbaldehyde

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-04-19 to 2000-05-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

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

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

LLNA not available at the time of testing.

Test material

Test material information

Constituent 1

Reference substance name:

8-isopropyl-6-methylbicyclo[2.2.2]oct-5-ene-2-carbaldehyde

EC Number:

267-308-7

EC Name:

8-isopropyl-6-methylbicyclo[2.2.2]oct-5-ene-2-carbaldehyde

Cas Number:

67845-30-1

Molecular formula:

C13H20O

IUPAC Name:

8-isopropyl-6-methylbicyclo[2.2.2]oct-5-ene-2-carbaldehyde

Constituent 2

Reference substance name:

7-isopropyl-5-methylbicyclo[2.2.2]oct-5-ene-2-carbaldehyde

EC Number:

299-795-7

EC Name:

7-isopropyl-5-methylbicyclo[2.2.2]oct-5-ene-2-carbaldehyde

Cas Number:

93904-56-4

Molecular formula:

C13H20O

IUPAC Name:

7-isopropyl-5-methylbicyclo[2.2.2]oct-5-ene-2-carbaldehyde

impurity 1

Reference substance name:

rel-(1S,2R,4R)-1-isopropyl-4-methylbicyclo[2.2.2]oct-5-ene-2-carbaldehyde

Cas Number:

36208-34-1

Molecular formula:

C13H20O

IUPAC Name:

rel-(1S,2R,4R)-1-isopropyl-4-methylbicyclo[2.2.2]oct-5-ene-2-carbaldehyde

Test material form:

liquid

Specific details on test material used for the study:

IdentificationL Lierral
Description: colourless to pale yellow liquid
Batch Number: 9000365675
Purity: 99.5%
Stability of test item: Stable under storage conditions; expiration date: 20-Dec-2000
Storage conditions: In the original container, at room temperature (range of 20 +/- 3 °C), away from direct sunlight

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Ibm: GOHI; SPF-quality guinea pigs (Himalayan spotted)

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: 4 - 6 weeks
- Weight at study initiation: 312 - 429 g
- Housing: Animals were housed individually in cages with standard softwood bedding
- Diet (e.g. ad libitum): Pelleted standard guinea pig breeding / maintenance diet, containing Vitamin C, ad libitum
- Water (e.g. ad libitum): Community tap water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 - 23 °C
- Humidity (%): 37 - 61 % (relative)
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): An automatically controlled light cycle of 12 hours light and 12 hours dark. Music was played during the light period.

IN-LIFE DATES: From: 2000-04-19 To: 2000-05-29

Study design: in vivo (non-LLNA)

No. of animals per dose:

10

Details on study design:

RANGE FINDING TESTS
Pre-tests performed before and during the acclimatisation period of the control and test group
- Intradermal injections
Four intradermal injections (0.1 mL/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of one guinea pig. One week later intradermal injections (0.1 mL/site) were made into the clipped flank of the same guinea pig at concentrations of 25, 50 and 100 % of the test material. Corn oil was used for the dilutions. Dermal reactions were assessed 24 hours later. Based on the results, the test material concentration of 50 % was selected for intradermal induction in the main study.
- Epidermal applications
Four intradermal injections (0.1 mL/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of two guinea pigs. One week later both flanks of each of the guinea pigs were clipped and shaved just prior to the application. Thereafter 4 patches of filter paper (3 x 3 cm) were saturated with the test material at 25, 50, 75 and 100 % and applied to the clipped and shaved flanks. Corn oil was used for the dilutions. The volume of test material preparation applied was approximately 0.2 mL. The patches were covered by a strip of aluminium foil and firmly secured by elastic plaster wrapped around the trunk and covered with impervious adhesive tape. This procedure ensured the intensive contact of the test material. The dressings were removed after an exposure period of 24 hours.
Twenty-one hours after removal of the dressing the application site was depilated in order to visualise any resulting erythema. The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema.
No highest non-irritating concentration could be determined. Therefore, a second pre-test was performed with two additional naive guinea pigs, treated in the same way as that described previously, with the concentrations of 1, 5, 10 and 15 % in corn oil, using an application volume of approximately 0.2 mL. Based on the results, the concentration selected for epidermal induction and challenge in the main study was 100 and 15 %, respectively.

MAIN STUDY
INDUCTION EXPOSURE
- Intradermal injections performed on test day 1
An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 mL/site) were made at the border of a 4 x 6 cm area in the clipped region.
Test Group
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) The test material, at 50 % in corn oil.
3) The test material at 50 % in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
Control Group
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) Corn oil
3) 1:1 (w/w) mixture of corn oil in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

- Epidermal applications performed on test day 8
One week after the injections, the scapular area (approximately 6 x 8 cm) was again clipped and shaved free of hair prior to the application. A 2 x 4 cm patch of filter paper was saturated with the undiluted test material and placed over the injection sites of the test animals. The volume of test material preparation applied was approximately 0.3 mL. The patch was covered with aluminium foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The occlusive dressings were left in place for 48 hours. The epidermal application procedure described ensures intensive contact of the test material.
The guinea pigs of the control group were treated as described above with corn oil only, also applied at a volume of approximately 0.3 mL.
The reaction sites were assessed 25 and 49 hours after removal of the bandage for erythema and oedema.

CHALLENGE EXPOSURE
- Performed on test day 22
The test and control guinea pigs were challenged two weeks after the epidermal induction application and were treated in the same way.
Hair was clipped and shaved from a 5 x 5 cm area on the left and right flank of each guinea pig just prior to the application. Two patches (3 x 3 cm) of filter paper were saturated with the test material at the highest non-irritating concentration of 15 % (left flank) and the vehicle only (corn oil applied to the right flank) using the same method as for the epidermal application. The volume of test material preparation and vehicle applied was approximately 0.2 mL. The dressings were left in place for 24 hours.
Twenty-one hours after removal of the dressing the test sites treated with the test material were depilated. The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema.

READINGS AND SCORING
The scoring system was performed by visual scoring of erythema, oedema and other clinical changes of skin conditions. They were assessed using the following Magnusson and Kligman grading scale:
0 = no visible change
1 = discrete or patchy erythema
2 = moderate and confluent erythema
3 = intense erythema and swelling
Grading of all animals was done by positioning the animal under true-light (Philips TLD 36W/84 or Osram 36W/31 830).

Challenge controls:

The control guinea pigs were challenged two weeks after the epidermal induction application and were treated in the same way as those in the test group.

Positive control substance(s):

yes

Remarks:

alpha-hexylcinnamaldehyde

Results and discussion

Positive control results:

None of the control and test animals showed skin reactions after the first challenge treatment with alpha-hexylcinnamaldehyde at 0.1 % (w/w) in PEG 400.
All test animals showed either moderate/confluent erythema at the 24 hour reading or discrete/patchy to moderate/confluent erythema at the 48 hour reading after the second challenge treatment with alpha-hexylcinnamaldehyde at 5 % in PEG 400. All control animals treated in the same way showed discrete/patchy erythema at the 24 and 48 hour reading.
Six and five out of 10 test animals were observed with discrete/patchy to moderate/confluent erythema at the 24 hour reading and discrete/patchy erythema at the 48 hour reading respectively after treatment with alpha-hexylcinnamaldehyde at 1 % in PEG 400. The control animals showed no skin reaction after the same treatment.

Therefore when applied at the non-irritating concentration of 1 % in PEG 400, alpha-hexylcinnamaldehyde was found to be a skin sensitiser.

In vivo (non-LLNA)

Results

Results 1

Reading:

other: Challenge

Hours after challenge:

24

Group:

test chemical

Dose level:

15 % in corn oil

No. with + reactions:

10

Total no. in group:

10

Remarks on result:

other: Reading: other: Challenge. . Hours after challenge: 24.0. Group: test group. Dose level: 15 % in corn oil. No with. + reactions: 10.0. Total no. in groups: 10.0.

Results 2

Reading:

other: Challenge

Hours after challenge:

48

Group:

test chemical

Dose level:

15 % in corn oil

No. with + reactions:

6

Total no. in group:

10

Remarks on result:

other: Reading: other: Challenge. . Hours after challenge: 48.0. Group: test group. Dose level: 15 % in corn oil. No with. + reactions: 6.0. Total no. in groups: 10.0.

Results 3

Reading:

other: Challenge

Hours after challenge:

24

Group:

test chemical

Dose level:

Corn oil vehicle only

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: other: Challenge. . Hours after challenge: 24.0. Group: test group. Dose level: Corn oil vehicle only. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 4

Reading:

other: Challange

Hours after challenge:

24

Group:

negative control

Dose level:

15 % in corn oil

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: other: Challange. . Hours after challenge: 24.0. Group: negative control. Dose level: 15 % in corn oil. No with. + reactions: 0.0. Total no. in groups: 5.0.

Results 5

Reading:

other: Challenge

Hours after challenge:

24

Group:

negative control

Dose level:

Corn oil vehicle only

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: other: Challenge. . Hours after challenge: 24.0. Group: negative control. Dose level: Corn oil vehicle only. No with. + reactions: 0.0. Total no. in groups: 5.0.

Any other information on results incl. tables

Main Study

Induction

- Skin effects after intradermal induction performed on test day 1

The expected and common findings were observed in the control and test group after the different applications using FCA intradermally. These findings consisted of erythema, oedema, necrotising dermatitis, encrustation and exfoliation of encrustation.

 

- Skin effects after epidermal induction performed on test day 8

Control group: No erythematous or oedematous reaction was observed in the animals treated with corn oil only.

Test group: Discrete/patchy erythema was observed in nine (at the 24 hour reading) and six (at the 48 hour reading) out of 10 animals after treatment with the undiluted test material.

 

Challenge

- Skin effects after the challenge performed on test day 22

Control group: No skin reactions were observed in the animals when treated with either corn oil only or when treated with the test material at 15 % in corn oil.

Test group: Discrete/patchy to moderate/confluent erythema were observed in all animals at the 24 hour reading and discrete/patchy erythema was observed in six out of 10 animals at the 48 hour reading after treatment with the test material at 15 % in corn oil. No skin reactions were observed in the animals when treated with corn oil only.

 

Other Observations

There were no deaths during the course of the study, hence no necropsies were performed. No signs of systemic toxicity were observed in the animals. The body weight of the animals was within the range commonly recorded for animals of this strain and age.

Table 1: Main Study Results

Test Group / Dose Level	Animal Number	Reaction Readings Following Bandage Removal
		24 Hours	48 Hours
Epidermal Induction
Control / Corn Oil	401 402 403 404 405	0 0 0 0 0	0 0 0 0 0
Test Group / 100 % Test Material	406 407 408 409 410 411 412 413 414 415	1 1 1 0 1 1 1 1 1 1	1 0 1 0 1 1 0 1 1 0
Challenge
Control / Corn Oil	401 402 403 404 405	0 0 0 0 0	0 0 0 0 0
Control / 15 % Test Material	401 402 403 404 405	0 0 0 0 0	0 0 0 0 0
Test Group / Corn oil	406 407 408 409 410 411 412 413 414 415	0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0 0 0 0 0
Test Group / 15 % Test Material	406 407 408 409 410 411 412 413 414 415	1 1 2 1 1 2 1 1 2 2	0 1 1 1 0 1 0 0 1 1

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Under the conditions of this study, the test material was determined to be sensitiser in male albino guinea pigs.

Executive summary:

The potential of the test material to cause skin sensitisation was assessed under GLP conditions and in accordance with the standardised guidelines OECD 406 and EU Method B.6.

In order to assess the cutaneous allergenic potential of the test material, the maximisation test was performed in 15 (10 test and 5 control) male albino guinea pigs (Ibm: GOHI; SPF-quality guinea pigs (Himalayan spotted)).

The intradermal induction of sensitisation in the test group was performed in the nuchal region with a 50 % dilution of the test material in corn oil and in an emulsion of Freund's Complete Adjuvant (FCA) / physiological saline. The epidermal induction of sensitisation was conducted for 48 hours under occlusion with the undiluted test material one week after the intradermal induction. The animals of the control group were intradermally induced with corn oil and FCA/physiological saline and epidermally induced with corn oil under occlusion.

Two weeks after epidermal induction, the control and test animals were challenged by epidermal application of the test material at 15 % in corn oil and corn oil alone under occlusive dressing.

Cutaneous reactions were evaluated at 24 and 48 hours after removal of the dressing. No toxic symptoms were evident in the guinea pigs of the control or test group. No deaths occurred.

All ten test animals showed discrete/patchy to moderate/confluent erythema at the 24 hour reading after the challenge treatment at 15 % (w/w) in corn oil. Discrete/ patchy erythema was observed in six out of ten animals at the 48 hour reading. No skin effect was observed in the control group.

Under the conditions of this study, the test material was determined to be sensitiser in male albino guinea pigs.