Reaction mass of (1RS,2RS,7SR,8RS,9E)-9-Ethylidene-3-oxatricyclo [6.2.1.0(2,7)] undecane and (1RS,2RS,7SR,8RS,9Z)-9-Ethylidene-3-oxatricyclo [6.2.1.0(2,7)] undecane and (1RS,2SR,7SR,8SR,10E)-10-Ethylidene-3-oxatricyclo[6.2.1.0(2,7)] undecane and (1RS,2SR,7SR,8SR,10Z)-10-Ethylidene-3-oxatricyclo [6.2.1.0(2,7)] undecane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (1RS,2RS,7SR,8RS,9E)-9-Ethylidene-3-oxatricyclo [6.2.1.0(2,7)] undecane and (1RS,2RS,7SR,8RS,9Z)-9-Ethylidene-3-oxatricyclo [6.2.1.0(2,7)] undecane and (1RS,2SR,7SR,8SR,10E)-10-Ethylidene-3-oxatricyclo[6.2.1.0(2,7)] undecane and (1RS,2SR,7SR,8SR,10Z)-10-Ethylidene-3-oxatricyclo [6.2.1.0(2,7)] undecane

Molecular formula:

C12H18O

IUPAC Name:

Reaction mass of (1RS,2RS,7SR,8RS,9E)-9-Ethylidene-3-oxatricyclo [6.2.1.0(2,7)] undecane and (1RS,2RS,7SR,8RS,9Z)-9-Ethylidene-3-oxatricyclo [6.2.1.0(2,7)] undecane and (1RS,2SR,7SR,8SR,10E)-10-Ethylidene-3-oxatricyclo[6.2.1.0(2,7)] undecane and (1RS,2SR,7SR,8SR,10Z)-10-Ethylidene-3-oxatricyclo [6.2.1.0(2,7)] undecane

Test material form:

gas under pressure: refrigerated liquefied gas

Details on test material:

- Physical state: liquid
- Storage condition of test material: In refrigerator (2-8°C) in the dark under nitrogen

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J strain, inbred, SPF-Quality

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Recognised animal supplier.
- Age at study initiation: approximately 9 months old
- Weight at study initiation: 22 - 25 g
- Housing: Animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material
- Diet (e.g. ad libitum): Certified pellet diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): 15
- Photoperiod: 12 hours light / 12 hours dark

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Preliminary test: 50, 100%
Main study: 0, 25, 50 and 100%

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
A pre-screen test was conducted in order to select the highest test substance concentration to be used in the main study. Two test substance concentrations were tested; a 100% and 50% concentration. The test system, procedures and techniques were identical to those used in the main study except that assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected (in the range of 8 to14 weeks old). Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6.

MAIN STUDY
Three groups of five animals were treated with one test substance concentration (0, 25, 50 or 100 %) per group. The highest test substance concentration was selected from the pre-screen test. One group of five animals was treated with vehicle.

- Induction: The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance concentration, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test substance.
- Node excision: Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 μCi of 3H-methyl thymidine. After approximately five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) with Euthasol® 20%. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.
- Tissue processing and radioactivity measurements: A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) and stored in the refrigerator until the next day. Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

- Observations:
Mortality/Viability: Twice daily.
Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing) according to the following numerical scoring system. Furthermore, a description of all other (local) effects was recorded.

Positive control substance(s):

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

Results and discussion

Positive control results:

In a separate 'positive control study' performed according to OECD 429 during April 2013, the sensitivity of the strain of mouse used in this study was assessed using the known sensitiser, hexylcinnamaldehyde. The positive control was tested at concentrations of 5, 10 and 25% v/v in the same vehicle. The highest concentration tested showed a Stimulation Index (SI) of 7.7 and met the criteria for a 'positive' result.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see below

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see below

Any other information on results incl. tables

In the preliminary screening test,very slight erythema was noted in both animals at 100% during Days 2-5. Scaliness was noted in these animals on Day 6. No signs of systemic toxicity were observed in any of the animals examined. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Based on these results, the highest test substance concentration selected for the main study was a 100% concentration. In the main test, there were no deaths or signs of systemic toxicity, and body weights were unaffected.The slight irritation of the ears as shown by all animals treated at 100% between Days 2 and 5, and the scalines noted in four females at 100% on Day 6 was considered not to have a toxicologically significant effect on the activity of the nodes.The radioactive disintegrations per minute (dpm) and stimulation index (SI) are given in the table below. The data showed a dose-response and an EC3 value (the estimated test substance concentration that will give a SI =3) of 35% was calculated. 

Table 1. Results from the definitive test

group	TS1	animal	Size nodes2		DPM3/ animal	mean			mean
	(%)		left	right		DPM ± SEM4			SI ± SEM
1	0	1	n	n	428	547	±	70	1	±	0.2
		2	n	n	435
		3	n	n	691
		4	n	n	743
		5	n	n	440
2	25	6	n	n	1166	1427	±	335	2.6	±	0.7
		7	+	+	1968
		8	+	+	903
		9	N	n	661
		10	+	+	2435
3	50	11	+	+	1951	1971	±	456	3.6	±	1
		12	+	+	1884
		13	n	n	1400
		14	+	+	3649
		15	n	n	971
4	100	16	++	++	2998	3205	±	283	5.9	±	0.9
		17	++	++	2598
		18	++	++	3814
		19	++	++	2675
		20	++	++	3942

 1. TS = test substance (% w/w).

2. Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +,++ or +++: degree of enlargement, n: considered to be normal).

3. DPM= Disintegrations per minute 

 

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study the test material is considered to be sensitising to skin with EC3 of 35%.

Executive summary:

The study was performed to assess the skin sensitisation potential of the test material in the CBA/J strain, inbred, SPF-Quality mouse following topical application to the dorsal surface of the ear. The study was performed to GLP and the method followed OECD 429. In a preliminary screening test mice were treated by daily application of 25 μl of the undiluted test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mice was observed twice daily and local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6. Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.  Based on the preliminary test, the concentrations selected for the main test were 0%, 25%, 50% and 100% v/v. Groups of five mice were treated with the undiluted test item or the test item at concentrations of 50% or 25% v/v in acetone/olive oil 4:1. The mice were treated by daily application of 25 µl of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). A further group of five mice received the vehicle alone in the same manner. All animals were observed twice daily for mortality and once daily on days 1-6 (on days 1-3 within 1 hour after dosing) for clinical signs. Any signs of toxicity or signs of ill health during the test were recorded. The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination). At test termination, five hours after administration of 3HTdR, the test organisms were humanly euthanized. The draining auricular lymph nodes from the five mice were excised and pooled for each experiment group. Following appropriate preparation,3HTdR incorporation was measured by β-scintillation counting and the number of radioactive disintegrations per minute (dpm) was measured. The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute and as the ratio of3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes. The substance was regarded as sensititising if at least one concentration of the substance resulted in a 3-fold or greater increase in3HTdR incorporation compared to control values. Any substance failing to produce a 3-fold or greater increase in3HTdR incorporation was classified as non-sensitising. In the main test, there were no deaths or signs of systemic toxicity, and body weights were unaffected. A stimulation index (SI) of more than 3 was noted at the 50% and 100% test material concentrations; the data showed a dose-response and an EC3 value (the estimated test substance concentration that will give a SI =3) of 35% was calculated. Accordingly, the test material was considered to be sensitising under the conditions of the test.