Bicyclo[2.2.1]heptane, 2-ethyl-5-methoxy-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 17 May 1988 and 23 June 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

guideline not stated

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

An LLNA is not performed because other reliable information is available.

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

403-610-9

EC Name:

-

Cas Number:

122795-41-9

IUPAC Name:

1-ethyl-3-methoxytricyclo[2.2.1.0²,⁶]heptane; 2-ethyl-5-methoxybicyclo[2.2.1]heptane; 2-ethyl-6-methoxybicyclo[2.2.1]heptane

Test material form:

liquid

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: A reputable supplier. For full details please see full study report
- Weight at study initiation: 300 - 700 g
- Housing: Guinea pigs were housed individually in 1/2" stainless steel wire mesh cages. Size in accordance with "Guide for the Care and Use of Laboratory Animals" of the Institute of Laboratory Resources, National Research Council
- Diet: Wayne Guinea pig DietR, ad libitum
- Water: ad libitum
- Acclimation period: Minimum five (5) days.

ENVIRONMENTAL CONDITIONS
- Temperature: maintained at a temperature of 22°C ± 3°C:
- Humidity: 30 to 70%.
- Photoperiod: 12 hours light, 12 hours dark

Study design: in vivo (non-LLNA)

Induction

Route:

epicutaneous, semiocclusive

Vehicle:

other: Ethanol (80%)

Concentration / amount:

2%

Day(s)/duration:

3 times 6 hours

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Challenge

Route:

epicutaneous, semiocclusive

Vehicle:

other: Ethanol (80%)

Concentration / amount:

2%

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Dose-range - four (4)
Test article - twenty (20)
Positive control - five (5)
Negative control - ten (10)
Naive challenge - four (4)
Naive rechallenge - four (4)*
*Four animals from the negative control group were utilized as naive control animals for rechallenge.

Details on study design:

RANGE FINDING TESTS:
Prior to initiation of the study, the irritation potential was determined. Six (6) unexposed animals (3 males and 3 females) were each exposed to different concentrations of the test material by the technique described in site preparation and treatment. In this test, both sides of the animal were shaved and exposed to one concentration of the material. The procedure described in Table 1 for primary challenge was used in grading of the response except that only 24 hour grades were obtained. The dose chosen for induction, challenge and rechallenge was 2. 0%.

MAIN STUDY
- Site preparation and treatment:
The left shoulder of each animal was clipped free of hair with an Oster Model A2 clipper twenty-four hours prior to the 1st, 2nd and 3rd applications of the test material. The shaved area was approximately 5 x 10 cm, i.e. 10% of the body surface. The test material was applied to 20 guinea pigs (10 male and 10 female). The material was applied beneath a 37 x4 0 mm Parke-Davis Readi-Bandage , Parke-Davis and Company, Greenwood, South Carolina) and covered with a dental dam. The patch and dam were held in place with clips attached to the sides of the guinea pig restrainer. The patches were allowed to remain in place for six hours, after which the rubber dams and patches were removed. The treated sites were examined after each dosing day and scored at 24 and 48 hours according to Table 1. This procedure was performed once a week for three weeks, a total of three six-hour inductions. The initial body weight of the animals was recorded. An additional group of ten guinea pigs (5 males and 5 females) was treated with vehicle (Ethanol 80%). Fourteen days after the last induction sensitizing exposure, the animals were challenged in the same manner on a naive site on the left side. Four naive pigs (2 males and 2 females) were induced with the test material, 88-206-01, at a 2.0% concentration, on challenge day in the same manner. Seven days after the primary challenge, the experimental group and a group of four naive pigs were rechallenged on the right side with the test article at a 2.0% concentration.

Four animals from the negative control group (2 males and 2 females) were utilized as naive controls for rechallenge. Local and systemic effects were observed. Twenty-four hours after the challenge and rechallenge, all animals were depilated with Neet Cream Hair Remover (Whitehall Laboratories, Inc. New York 10017). The depilatory was placed on the test sites and surrounding areas and left for no more than 30 minutes. After this time, the animals were thoroughly rinsed with water, gently patted dry and returned to their cages. A minimum of two hours after depilation test sites were graded. The grading was repeated 24 hours later (48 hour grade).

- Frequency of applications:
Three (3) inductions: 88-206-01 (2.0%) (experimental animals)
Three (3) inductions: DNCB (0.3%) (positive control animals)
Three (3) inductions: Ethanol (80%) (negative control animals)
One (1) challenge: 88-206-01 (2.0%) (experimental animals)
One (1) challenge: DNCB (0.3%) (positive control animals)
One (1) challenge: Ethanol (80%) (negative control animals)
One ( 1) challenge: 88-206-01 (2.0%) (naive animals)
One (1) rechallenge: 88-206-01 ( 2.0%) (experimental animals)
One (1) rechallenge: 88- 206-01 ( 2.0%) (naive animals)

Challenge controls:

A concurrent positive control group consisting of five guinea pigs (3 males and 2 females) was treated with (0.3%) 1-chloro- 2 , 4-dinitrobenzene in 80% ethanol (80:20 ethanol/distilled water).

Positive control substance(s):

yes

Remarks:

1-chloro-2,4 dinitrobenzene (DNCB)

Results and discussion

Positive control results:

A positive response was elicited in the animals receiving the positive control article, 1-chloro-2,4 dinitrobenzene (DNCB)

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

2.0%

No. with + reactions:

2

Total no. in group:

20

Clinical observations:

2 animals with score [1] 1 = slight or confluent or moderate patchy erythema

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

2.0%

No. with + reactions:

3

Total no. in group:

20

Clinical observations:

3 animals with score [1] 1 = slight or confluent or moderate patchy erythema

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

other: naive animals

Dose level:

2.0%

No. with + reactions:

0

Total no. in group:

4

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

other: naive animals

Dose level:

2.0%

No. with + reactions:

0

Total no. in group:

4

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

DNCB (0.3%)

No. with + reactions:

5

Total no. in group:

5

Clinical observations:

All animals had score of [3] 3 = severe erythema with/without edema

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

DNCB (0.3%)

No. with + reactions:

5

Total no. in group:

5

Clinical observations:

All animals had score of [3] 3 = severe erythema with/without edema

Results 7

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

Ethanol (80%)

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: Ethanol (80%). No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 8

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

Ethanol (80%)

No. with + reactions:

0

Total no. in group:

10

Results 9

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

88-206-01 (2.0%)

No. with + reactions:

0

Total no. in group:

20

Results 10

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

88-206-01 (2.0%)

No. with + reactions:

0

Total no. in group:

20

Results 11

Reading:

rechallenge

Hours after challenge:

24

Group:

other: naive animals

Dose level:

88-206-01 (2.0%)

No. with + reactions:

0

Total no. in group:

4

Results 12

Reading:

rechallenge

Hours after challenge:

48

Group:

other: naive animals

Dose level:

88-206-01 (2.0%)

No. with + reactions:

0

Total no. in group:

4

Any other information on results incl. tables

Results of dose range finding study:

Animal no	Sex	24 hour scores
		Site 1	Site 2	Site 3	Site 4
1	♂	0	3	3	0
2	♂	0	3	3	0
3	♀	0	3	3	0
4	♀	0	3	3	0

Site 1 = 1.0%

Site 2 = 10%

Site 3 = 25%

Site 4 = 100%

Animal no	Sex	24 hour scores
		Site 1	Site 2	Site 3	Site 4
1	♂	0	0	0	0
2	♀	0	0	0	3

Site 1 = 1.0%

Site 2 = 2.5%

Site 3 = 5.0%

Site 4 = 7.5%

Challenge A positive response was elicited in the animals receiving the positive control article, 1-chloro-2,4 dinitrobenzene (DNCB). No responses were observed in the animals treated with 80% ETOH. No responses were observed in the naive group at 24 or 48 hours after challenge. Two positive responses were observed at 24 hours and three positive responses were observed at 48 hours after challenge in the experimental animals receiving the test article at a 2.0% concentration. Rechallenge Responses were observed in the naive animals at 24 and 48 hours after rechallenge. No positive responses were observed in the experimental animals at 24 or 48 hours after rechallenge.

Applicant's summary and conclusion

Interpretation of results:

other: Not sensitising according to EU CLP (EC 1272/2008 and its amendments).

Conclusions:

Based upon the observations made in the Delayed Contact Hypersensitivity Study, test article, induced, challenged and rechallenged at a 2.0% concentration did not cause delayed contact hypersensitivity in guinea pigs.

Executive summary:

The sensitising potential of the test substance was tested according to OECD TG 406, GPMT test. In preliminary dose-range-finding studies to determine the highest non irritating dose, six animals (three males and three females) were each exposed to four concentrations of BB-206-01 (Neoproxen). Based upon the results of the dose-range-finding studies the dose chosen for induction, challenge and rechallenge was 2.0%.

In order to determine if the test article is capable of causing delayed contact hypersensitivity, BB-206-01 was dermally applied to twenty guinea pigs (ten males and ten females) for a total of three six-hour insult periods at a 2.0% concentration.

A positive control was also included: A group of five guinea pigs (three males and two females) was treated with 1-chloro-2, 4 dinitrobenzene (DNCB) at a 0.3% concentration for a total of three six-hour insult periods. An additional group of ten guinea pigs (five males and five females) was treated with vehicle (ethanol-80% [ETOH]) for a total of three six-hour insult periods.

Fourteen days after the last induction period, all animals were challenged at a naive site. A positive response was elicited in the animals receiving the positive control article, l-chloro-2, 4 dinitrobenzene (DNCB). No responses were observed in the animals treated with 80% ETOH. No responses were observed in the naive group at 24 or 48 hours after challenge. Two positive responses were observed at 24 hours and three positive responses were observed at 48 hours after challenge in the experimental animals receiving the test article at a 2.0% concentration.

The experimental animals were rechallenged seven days after challenge, with the test article at a 2.0% concentration. Four animals from the negative control group (2 males and 2 females) served as naive control animals for the rechallenge. Responses were observed in the naive control animals at 24 and 48 hours after rechallenge, being indicative for irritation. No positive responses were observed in the experimental animals at 24 or 48 hours after rechallenge.

Based upon the observations made in the Delayed Contact Hypersensitivity Study, test article, BB-206-01 (Neoproxen) induced, challenged and rechallenged at a 2.0% concentration did not cause delayed contact hypersensitivity in guinea pigs.

It can be argued that the concentration (2%) used for induction and challenge is quite low, though some slight irritation was observed during rechallange in the naive control group animals. Neoproxen as such does not contain skin sensitizing structural alerts as can be seen when running the OECD Toolbox and/or Derek Nexus. This further supports the non-skin sensitizing potential.