[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04-09-2013 to 23-09-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

Justification for type of information:

Information as to the availability of the in vivo study is provided in 'attached justification'.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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 (incl. QA statement)

Remarks:

inspected: March 2013 ; signature: May 2013

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (4RS,4aRS,8RS,8aRS)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one and (4RS,4aSR,8SR,8aRS)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one and (4RS,4aSR,8SR,8aSR)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one

Molecular formula:

C13H22O

IUPAC Name:

Reaction mass of (4RS,4aRS,8RS,8aRS)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one and (4RS,4aSR,8SR,8aRS)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one and (4RS,4aSR,8SR,8aSR)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one

Test material form:

liquid

Specific details on test material used for the study:

- Physical state: Liquid
- Storage condition of test material: In refrigerator (2-8°C) in the dark
- Other: clear colourless liquid

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 weeks old
- Weight at study initiation: 19- 23 grams; Body weight variation was within +/- 20% of the sex mean.
- Housing: Group housed, in labelled Makrolon cages sterilised sawdust as bedding material, paper and shelters as cage enrichment. On Day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage enrichment.
- Diet: Free access to pelleted rodent diet (certified, recognised supplier)
- Water: mains tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0
- Humidity (%): 40 to 70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: From: 04-09-2013 To: to 23-09-2013

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

- Preliminary test: Initially, two test substance concentrations were tested; at 50% and 100% concentration. The highest concentration was the maximum concentration as required in the test guidelines (undiluted for liquids). At a 50% test substance concentration, both ears showed very slight erythema of both animals. Days 3 and 4, and of one animal on Day 5 . At a 100% test substance concentration, both animals showed well-defined erythema on both ears on Day 3 and very slight erythema on Day 4. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values at both 50 and 100%. No mortality occurred and no signs of systemic toxicity were observed in any of the animals examined. The slight body weight loss noted for one animal (#3) at 100% and one animal (#2) at 50% was
considered not toxicologically significant since this change was minor in nature. Based on the pre-screen results, the highest test substance concentration selected for the main study was 100% concentration.
- Main test: 0% (vehicle control), 25, 50 and 100 %. Test concentrations were determined from the results of the preliminary test.

No. of animals per dose:

Preliminary test: Two per concentration: 50% and 100%.
Main test: 5 mice per dose group 0% (vehicle control), 25%, 50% and 100%

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. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation at most (maximum grade 2 and/or an increase in ear thickness < 25%) and is the highest possible concentration that can technically be applied. Two test substance concentrations were tested; a 50% and 100% concentration. The highest concentration was the maximum concentration as required in the test guidelines (undiluted for liquids). The test system, procedures and techniques were identical to those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Ear thickness measurements were conducted using a digital thickness gauge on prior to dosing on Day 1, 1-hour post application of test item on Days 1, 2 and 3 and on Days 4, 5 and 6.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: Following excision of the nodes. The individual SI is the ratio of the DPM/animal compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:
Three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the pre-screen test. One group of five animals was treated with vehicle (and a further group for the additional dose as control).
- 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 terminated 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 scintillation counter. 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.
- Bodyweights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
- Clinical Observations: 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 immediately after dosing). Numerical system documented in the full study report. Furthermore, a description of all other (local) effects was recorded.
- Ear Thickness: Measurements were conducted in the pre-screen test and in the main study. A digital thickness gauge was used to measure the ear thickness of each ear prior to dosing on Day 1, 1-hour post application of test substance on Days 1, 2 and 3 and on Days 4, 5 and 6 in order to monitor for any changes in ear thickness.

Positive control substance(s):

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

Statistics:

Analysis was conducted according to Basketter DA et al., A comparison of statistical approaches to the derivation of EC3 values from local lymph node assay dose responses. J Appl Toxicol 1999;19:261-266.

Results and discussion

Positive control results:

In a separate 'positive control study' performed according to OECD TG 429 during April 2013. the sensitivity of the strain of mouse used in this study was assessed using the known sensitiser, α-hexylcinnamaldehyde. The positive control was tested at concentrations 5, 10 and 25% in Acetone/Olive oil (4:1 v/v). The highest concentration tested showed a Stimulation Index (SI) of 7.7 ± 0.9 (at 25% v/v) and met the criteria for a 'positive' result. An EC3 value of 13.4% was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 16.0, 11.9, 16.9, 14.4, 16.5, 14.5 and 16.5%.

In vivo (LLNA)

Results

Results 1

Parameter:

EC3

Remarks:

%

Value:

60.5

Remarks on result:

other: See table below

Results 2

Parameter:

SI

Value:

1.9

Variability:

± 0.3

Test group / Remarks:

25% in acetone:olive oil (4:1)

Results 3

Parameter:

SI

Value:

2.6

Variability:

± 0.6

Test group / Remarks:

50% in acetone:olive oil (4:1)

Results 4

Parameter:

SI

Value:

4.5

Variability:

± 0.9

Test group / Remarks:

100%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: See tables.

DETAILS ON STIMULATION INDEX CALCULATION: See tables.

EC3 CALCULATION: The EC3 was determined based on the linear interpolation.

CLINICAL OBSERVATIONS: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.

BODY WEIGHTS: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The slight body weight loss noted at 25% was considered not toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent.

Any other information on results incl. tables

In the preliminary screening test: at 50% and 100% concentration. The highest concentration was the maximum concentration as required in the test guidelines (undiluted for liquids).At a 50% test substance concentration, both ears showed very slight erythema of both animals. Days 3 and 4, and of one animal on Day 5 . At a 100% test substance concentration, both animals showed well-defined erythema on both ears on Day 3 and very slight erythema on Day 4. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values at both 50 and 100%. No mortality occurred and no signs of systemic toxicity were observed in any of the animals examined. The slight body weight loss noted for one animal (#3) at 100% and one animal (#2) at 50% was considered not toxicologically significant since this change was minor in nature. Based on the pre-screen results, the highest test substance concentration selected for the main study was 100% concentration.

 

In the main test: at50% test substance concentration, very slight erythema was noted for all animals on both ears between Days 2 and 5, and for four animals on one or both ears on Day 6. At a 100% test substance concentration, erythema was noted for all animals on both ears (well defined on Days 2 and 3 and very slight on Days 4, 5 and 6). Scales were noted on both ears of all animals treated at 100% on Day 6.

 

The radioactive disintegrations per minute (dpm) and stimulation index (SI) are given in the table below. The SI values calculated for the substance concentrations 25, 50 and 100% were 1.9, 2.6 and 4.5, respectively. The data showed a dose-response and an EC3 value (the estimated test substance concentration that will give a SI =3) of 60.5% was calculated.

Table 1. Results from the definitive test

Group	TS (%) #1	Number	Size nodes #2		DPM / animal #3	mean			mean
			left	right		DPM ± SEM #4			SI ± SEM
1	0	1	n	n	150	240	±	36	1.0	±	0.2
		2	n	n	164
		3	n	n	335
		4	n	n	299
		5	n	n	252
2	25	6	n	n	503	444	±	36	1.9	±	0.3
		7	n	n	452
		8	n	n	387
		9	n	n	341
		10	n	n	537
3	50	11	n	n	334	629	±	92	2.6	±	0.6
		12	+	+	822
		13	+	+	800
		14	n	n	677
		15	+	+	511
4	100	16	n	n	1098	1089	±	130	4.5	±	0.9
		17	+	+	1221
		18	+	+	1386
		19	+	+	610
		20	+	+	1130

#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

#4. SEM = Standard Error of the Mean

Applicant's summary and conclusion

Interpretation of results:

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

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study the test item is considered to be sensitising to skin with EC3 of 60.5%.

Executive summary:

The study was performed to OECD TG 429, EU Method B.42 and US EPA OPPTS 870.2600 guidelines under GLP to assess the skin sensitisation potential of the test material in the CBA/J strain mouse following topical application to the dorsal surface of the ear. In a preliminary screening test mice were treated by daily application of 25 μl of the test substance at 50% and 100% v/v in acetone/olive oil 4:1 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 on prior to dosing on Day 1, 1-hour post application of test item on Days 1, 2 and 3 and on Days 4, 5 and 6. Based on the pre-screen results, the highest test substance concentration selected for the main study was a 50% concentration. A mean ear thickness increase of equal to or greater than 25% and/or well-defined irritation at the most (maximum grade 2) was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation. No irritation or signs of systemic toxicity were observed. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Based on the preliminary test, the concentrations selected for the main test were 0%, 25%, 50% and 100% v/v. In the main test, three groups of five female CBA/J mice were treated with test substance concentrations of 25, 50 or 100% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 v/v)).At a 50% test substance concentration, very slight erythema was noted for all animals on both ears between Days 2 and 5, and for four animals on one or both ears on Day 6. At a 100% test substance concentration, erythema was noted for all animals on both ears (well-defined on Days 2 and 3 and very slight between Days 4 and 6). Scales were noted on both ears of all animals treated at 100% on Day 6.All auricular lymph nodes were considered normal in size, except for the nodes of the majority of animals treated at 50% and 100% which were enlarged. No macroscopic abnormalities of the surrounding area were noted in any of the animals in the vehicle control and treated groups. Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 444, 629 and 1089 DPM, respectively. The mean DPM/animal value for the vehicle control group was 240 DPM.The SI values calculated for the substance concentrations 25, 50 and 100% were 1.9, 2.6 and 4.5, respectively. These results indicate that the test substance could elicit an SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test substance concentration that will give a SI =3) of 60.5% was calculated. Under the conditions of this study, the test substance would be considered to be classified under Regulation (EC) No 1272/2008 as skin sensitizer category 1B.