5-ethylidene-8,9,10-trinorborn-2-ene, manufacture of, by-products from

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24-06-2020 to 08-07-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Cyclopentadiene

EC Number:

208-835-4

EC Name:

Cyclopentadiene

Cas Number:

542-92-7

Molecular formula:

C5H6

IUPAC Name:

cyclopenta-1,3-diene

Constituent 2

Reference substance name:

4-vinylcyclohexene

EC Number:

202-848-9

EC Name:

4-vinylcyclohexene

Cas Number:

100-40-3

Molecular formula:

C8H12

IUPAC Name:

4-vinylcyclohexene

Constituent 3

Reference substance name:

5-ethylidene-8,9,10-trinorborn-2-ene

EC Number:

240-347-7

EC Name:

5-ethylidene-8,9,10-trinorborn-2-ene

Cas Number:

16219-75-3

Molecular formula:

C9H12

IUPAC Name:

5-ethylidenebicyclo[2.2.1]hept-2-ene

Constituent 4

Reference substance name:

Cycloocta-1,5-diene

EC Number:

203-907-1

EC Name:

Cycloocta-1,5-diene

Cas Number:

111-78-4

Molecular formula:

C8H12

IUPAC Name:

cycloocta-1,5-diene

Constituent 5

Reference substance name:

3a,4,7,7a-tetrahydro-1H-indene

EC Number:

221-260-3

EC Name:

3a,4,7,7a-tetrahydro-1H-indene

Cas Number:

3048-65-5

Molecular formula:

C9H12

IUPAC Name:

3a,4,7,7a-tetrahydro-1H-indene

Constituent 6

Reference substance name:

5-methylbicyclo[2.2.1]hept-2-ene

EC Number:

212-509-7

EC Name:

5-methylbicyclo[2.2.1]hept-2-ene

Cas Number:

822-96-8

Molecular formula:

C8H12

IUPAC Name:

5-methylbicyclo[2.2.1]hept-2-ene

Constituent 7

Reference substance name:

3a,4,7,7a-tetrahydro-4,7-methanoindene

EC Number:

201-052-9

EC Name:

3a,4,7,7a-tetrahydro-4,7-methanoindene

Cas Number:

77-73-6

Molecular formula:

C10H12

IUPAC Name:

3a,4,7,7a-tetrahydro-4,7-methanoindene,

Constituent 8

Reference substance name:

3a,4,7,7a-tetrahydro-4-methyl-1H-indene

EC Number:

231-021-5

EC Name:

3a,4,7,7a-tetrahydro-4-methyl-1H-indene

Cas Number:

7413-02-7

Molecular formula:

C10H14

IUPAC Name:

4-methyl-3a,4,7,7a-tetrahydro-1H-indene

Constituent 9

Reference substance name:

Combination of: 6-(cyclohex-3-en-1-yl)bicyclo[2.2.1]hept-2-ene 2-vinyl-1,2,3,4,4a,5,8,8a-octahydro-1,4-methanonaphthalene 6-vinyl-1,4,4a,5,6,7,8,8a-octahydro-1,4-methanonaphthalene 2-vinyl-1,2,3,4,4a,5,8,8a-octahydro-1,4:5,8-dimethanonaphthalene 2,2'-bi(bicyclo[2.2.1]heptane-5,5'-diene) 3a,4,4a,5,8,8a,9,9a-octahydro-1H-5,8-methanocyclopenta[b]naphthalene 4,4a,4b,5,8,8a,9,9a-octahydro-1H-1,4-methanofluorene

Molecular formula:

C13H18 to C14H18

IUPAC Name:

Combination of: 6-(cyclohex-3-en-1-yl)bicyclo[2.2.1]hept-2-ene 2-vinyl-1,2,3,4,4a,5,8,8a-octahydro-1,4-methanonaphthalene 6-vinyl-1,4,4a,5,6,7,8,8a-octahydro-1,4-methanonaphthalene 2-vinyl-1,2,3,4,4a,5,8,8a-octahydro-1,4:5,8-dimethanonaphthalene 2,2'-bi(bicyclo[2.2.1]heptane-5,5'-diene) 3a,4,4a,5,8,8a,9,9a-octahydro-1H-5,8-methanocyclopenta[b]naphthalene 4,4a,4b,5,8,8a,9,9a-octahydro-1H-1,4-methanofluorene

Constituent 10

Reference substance name:

4,4a,4b,5,8,8a,9,9a-octahydro-1H-1,4-methanofluorene OR 4,4a,4b,5,8,8a,9,9a-octahydro-1H-1,4:5,8-dimethanofluorene

Molecular formula:

C14H18 to C15H18

IUPAC Name:

4,4a,4b,5,8,8a,9,9a-octahydro-1H-1,4-methanofluorene OR 4,4a,4b,5,8,8a,9,9a-octahydro-1H-1,4:5,8-dimethanofluorene

Test material form:

liquid

Details on test material:

Pale yellow liquid. Batch no: 776859. Storage room temperature in the dark.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V. Inc, Postbus 6174, 5960 AD Horst / The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 - 12 weeks
- Housing:
- Diet: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water: ad libitum tap water
- Acclimation period: 5 days minimum
- Indication of any skin lesions/Microbiological status of animals: Only animals without any visible signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°
- Humidity (%): 45-65
- Air changes (per hr): 8 minimum
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: To:

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

10, 25, 50%

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: Fully miscible up to 100% in vehicle
- Irritation: Two mice treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 50 and 100% once daily each on three consecutive days.
- Systemic toxicity: No, but irritation screened animals showed slight weight loss at 100% concentration
- Ear thickness measurements: Yes, 100% caused excessive thickening at 100% concentration.
- Erythema scores: 100% caused irritation beyond acceptable levels and eschar formation..

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index and that a dose response relationship is observed.

TREATMENT PREPARATION AND ADMINISTRATION:Each test group treated by topical application to the dorsal surface of each ear with test item (25 µL/ear/day spread over entire surface (~8 mm dia) for 3 consecutive days. A further group of mice were treated with vehicle alone. Five days after the first application (day 6) 250 µL of phosphate buffered saline containing 20 µCi of 3H-methyl thymidine (equivalent to 79.8 µCi/mL 3HTdR) were injected into tail vein. Mice sacrificed 5 hours later and lymph nodes harvested.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity.

Results and discussion

Positive control results:

EC3 = 12.5%. Historic control range 5.7 - 17.6%.

In vivo (LLNA)

Results

Results 1

Parameter:

EC3

Remarks on result:

not determinable

Remarks:

The EC3 value could not be calculated, since all S.I.´s up to the maximum tolerable concentration of 50% were below the threshold value of 3.

Results 2

Parameter:

SI

Value:

1.12

Test group / Remarks:

10% concentration

Results 3

Parameter:

SI

Value:

1.31

Test group / Remarks:

25% concentration

Results 4

Parameter:

SI

Value:

2.67

Test group / Remarks:

50% concentration

Remarks on result:

other: maximum tolerated concentration that does not cause irritation

Cellular proliferation data / Observations:

CLINICAL OBSERVATIONS: Top dose group showed slight to well defined ear erythema in all animals by day 3/4 which completely reversed by the day of sacrifice (day 6). No effects seen in any other dose group

BODY WEIGHTS: No adverse effects noted.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Not a skin sensitiser

Executive summary:

In a guideline mouse LLNA study, the test substance was not found to meet the criteria to be considered as a skin sensitiser when tested up to the maximum tolerated concentration. The study was performed using test item concentrations of 10, 25, and 50%. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment. The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. The animals treated with 50% test item concentration showed a very slight to well defined erythema of the ear skin (Score 1 to 2) and one animal showed scaly ears although this reversed after a few days of non-treatment.  The animals treated with 10 and 25% test item concentration did not show any signs of local irritation. In this study Stimulation Indices (S.I.) of 1.12, 1.31, and 2.67 were determined with the test item at concentrations of 10, 25, and 50% in acetone/olive oil (4+1 v/v), respectively. As an SI of 3 was not reached, the substance is not considered as a skin sensitiser.