Indeno[4,3a-b]furan, decahydro-2,2,7,7,8,9,9-heptamethyl-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 June 2003 to 01 July 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

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:

-

EC Number:

449-360-4

EC Name:

-

Cas Number:

647828-16-8

Molecular formula:

C18H32O

IUPAC Name:

decahydro-2,2,6,6,7,8,8-heptamethyl-2H-Indeno[4,5-b] furan

Test material form:

liquid

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca (CBA/Ca CruBR)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS:
- Source: Charles River UK Limited, Margate, Kent, UK
- Age at study initiation: 8-12 weeks old
- Weight at study initiation: 15 to 23 g
- Housing: Individually in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS:
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): was approximately 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours continuous light (06.00 to 18.00) and 12 hours darkness

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

100, 50 and 25 %

No. of animals per dose:

5

Details on study design:

ANIMAL ASSIGNMENT AND TREATMENT:
- Name of test method: Local Lymph Node Assay in the Mouse (LLNA) - individual animal approach
- Criteria used to consider a positive response: Stimulation Index (SI) of 3.0 or greater

TREATMENT PREPARATION AND ADMINISTRATION:
- Dose selection: Available information suggested that the test substance would not produce systemic toxicity or excessive local irritation at the highest suitable concentration.
- Preparation of Test Substance: The test substance was used undiluted and freshly prepared in acetone/olive oil 4:1.
- Choice of vehicle: Acetone/olive oil 4:1 produced the highest concentration that was suitable for dosing.
- Analysis of Test Substance: Determination, by analysis, of the concentration, homogeneity and stability of the test substance preparations was not appropriate because it was not specified in the Study Plan and is not a requirement of the Test Guideline.
- Test Substance Administration: The mice were treated by daily application of 25 µL of the appropriate concentration of the test substance or the vehicle alone to the dorsal surface of each ear for 3 consecutive days (Days 1, 2, 3). The test substance formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
- 3H-Methyl Thymidine Administration: Five days following the first topical application of the test substance (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline containing 3H-methyl thymidine 3HTdR:80µCi/mL, specific activity 2.0 Ci/mmol, giving a total of 20 µCi to each mouse.

Positive control substance(s):

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

Statistics:

Data were processed to give group mean values for dpm and standard deviations where appropriate. Individual and group mean dpm values were assessed for dose response relationships by linear regression analysis followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous pair wise comparisons were conducted using Dunnett's test.

Results and discussion

Positive control results:

The Stimulation Index (SI) expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group were determined to be 2.0 at 5 % v/v, 1.9 at 10 % v/v, and 6.8 at 25 % v/v. Therefore, alpha-Hexylcinnamaldehyde, Tech 85 % was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Value:

43.44

Results 2

Parameter:

SI

Value:

1.35

Remarks on result:

other: 25 %

Results 3

Parameter:

SI

Value:

3.18

Remarks on result:

other: 50 %

Results 4

Parameter:

SI

Value:

6.32

Remarks on result:

other: 100%

Any other information on results incl. tables

Clinical Observations and Mortality:

No mortality or signs of systemic toxicity were noted in the test or control animals during the study. Individual clinical observations and mortality data for test and control animals are given In Table 2.

Body Weight:

Body weight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period. Individual body weights and body weight changes for test and control animals are given in Table 3.

EC3 Value:

An EC3 value of 43.44 % was calculated using this equation: EC3 = c + [[(3 -d)/(b-d)] x (a-c)] (with a = lowest concentration giving stimulation index >3, b = actual stimulation index caused by 'a', c = highest concentration failing to produce a stimulation index of 3, and d = actual stimulation index caused by 'c')

Table 1. Individual DPMs and Stimulation Index (SI).

Concentration (% v/v) in Acetone/Olive Oil 4:1	Animal Number	dpm/ Animal^a	Mean dpm/Animal (Standard Deviation)	Stimulation Index (SI)^b	Result
Vehicle	1-1	1924.82	1766.62 (±346.82)	N/A	N/A
	1-2	2250.40
	1-3	1390.26
	1-4	1784.06
	1-5	1483.57
25	2-1	1312.38	2377.93 (±1293.63)	1.35	Negative
	2-2	1499.26
	2-3	2378.80
	2-4	2145.82
	2-5	4553.37
50	3-1	4415.68	5623.16** (±1162.54)	3.18	Positive
	3-2	5859.02
	3-3	4412.52
	3-4	6865.30
	3-5	6563.29
100	4-1	7027.93	11157.90* (±3228.46)	6.32	Positive
	4-2	13472.95
	4-3	14747.63
	4-4	11822.04
	4-5	8718.93

a = Total number of lymph nodes per animal is 2

b = Stimulation Index of 3.0 or greater indicates a positive result

N/A = Not applicable

* = Significantly different from control group p<0.05

** = Significantly different from control group p<0.01

Table 2. Individual Clinical Observations and Mortality Data.

Concentration (% w/w) in acetone/olive oil 4:1	Animal Number	Day 1		Day 2		Day 3		Day 4	Day 5	Day 6
		Pre-dose	1 hr post dose	Pre-dose	1 hr post dose	Pre-dose	1 hr post dose
Vehicle	1-1	0	0	0	0	0	0	0	0	0
	1-2	0	0	0	0	0	0	0	0	0
	1-3	0	0	0	0	0	0	0	0	0
	1-4	0	0	0	0	0	0	0	0	0
	1-5	0	0	0	0	0	0	0	0	0
25	2-1	0	0	0	0	0	0	0	0	0
	2-2	0	0	0	0	0	0	0	0	0
	2-3	0	0	0	0	0	0	0	0	0
	2-4	0	0	0	0	0	0	0	0	0
	2-5	0	0	0	0	0	0	0	0	0
50	3-1	0	0	0	0	0	0	0	0	0
	3-2	0	0	0	0	0	0	0	0	0
	3-3	0	0	0	0	0	0	0	0	0
	3-4	0	0	0	0	0	0	0	0	0
	3-5	0	0	0	0	0	0	0	0	0
100	4-1	0	0	0	0	0	0	0	0	0
	4-2	0	0	0	0	0	0	0	0	0
	4-3	0	0	0	0	0	0	0	0	0
	4-4	0	0	0	0	0	0	0	0	0
	4-5	0	0	0	0	0	0	0	0	0

Hr = Hour

0 = No signs of systemic toxicity

Table 3. Individual Body Weights and Body Weight Changes.

Concentration (% w/w) in acetone/olive oil 4:1	Animal Number	Body Weight Change (g)		Body Weight Change (g)
		Day 1	Day 6
Vehicle	1-1	19	20	1
	1-2	20	21	1
	1-3	18	18	0
	1-4	17	19	2
	1-5	18	19	1
25	2-1	18	19	1
	2-2	17	19	2
	2-3	18	19	1
	2-4	21	21	0
	2-5	17	17	0
50	3-1	18	19	1
	3-2	19	20	1
	3-3	18	19	1
	3-4	17	18	1
	3-5	19	19	0
100	4-1	19	21	2
	4-2	18	18	0
	4-3	20	20	0
	4-4	18	19	1
	4-5	20	20	0

Applicant's summary and conclusion

Interpretation of results:

other: Substance is a skin sensitiser (1B) according to EU CLP (1272/2008)

Conclusions:

The substance is a skin sensitiser (1B) in the LLNA test, OECD guideline 429.

Executive summary:

A Local Lymph Node Assay (LLNA) was performed in accordance with OECD TG 429 and under GLP conditions to assess the skin sensitisation potential of the test substance in the CBA/Ca strain mice following topical application to the dorsal surface of the ear. Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of the undiluted test material (100 %) and the test substance as a solution in acetone/olive oil 4:1 at concentrations of 50 % and 25 % v/v. A further group of five animals was treated with acetone/olive oil 4:1 alone. The Stimulation Index (SI) expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group were 1.35, 3.18, and 6.32 for the 25 %, 50 %, and 100 % exposure groups, respectively. The calculated EC3 value was 43.44 %. Based on these results, the test substance was considered to be a sensitiser (1B) under the conditions of the test.