Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
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:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected March 2013; signature: May 2013
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
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
- Other: clear colourless; pH (1% in water, indicative range) 8.5-8.3

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: approx. 10 weeks old
- Weight at study initiation: 18 - 25 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
- Water: mains tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): 10
- Photoperiod: 12 hours light / 12 hours dark

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
- Preliminary test: Two test substance concentrations were tested; at 100% and 50% concentration. The highest concentration was the maximum concentration as required in the test guidelines (undiluted for liquids).
- Main test: 0% (vehicle control), 25, 50 and 100% (undiluted). 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 the 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 100% and 50% 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 Days 1 (pre- and post-dosing), 2 (post-dosing), 3 (pre- and post-dosing), 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.

- 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 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 (pre- and post-dosing).
- Bodyweights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
- Clinical Observations: Twice daily (pre- and post-dosing) on Days 1-3. Once daily on Days 4-6 (on Days 1-3 between 3 and 4 hours after dosing).
- Irritation: Twice daily (pre- and post-dosing) on Days 1-3. Once daily on Days 4-6 (on Days 1-3 within 1 hour after dosing) according to the following numerical scoring system. Furthermore, a description of all other (local) effects will be recorded.
- Ear Thickness Based on the results of the pre-screen test no ear thickness measurements were conducted in the main study.
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 October 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 8.0 ± 1.8 (at 25% v/v) and met the criteria for a 'positive' result. An EC3 value of 14.1% was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 2 and 20%. The results of the 6 monthly HCA reliability checks of the recent years were 11.9, 16.9, 14.4, 16.5, 14.5 and 13.4%.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: See table below
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: See table below

Any other information on results incl. tables

In the preliminary screening test, Very slight erythema was scored for the animals in the 100% group on Days 1, 2 and 3 post-dosing however no signs of irritation were observed thereafter. 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 no irritation of ears was observed in any of the animals. One animal in the vehicle control group was excluded from interpretation, since this animals showed rales from Day 4 onwards in combination with a clear body weight loss of 2 grams. Based on available data it was considered that the study outcome was not adversely affected. Body weights and body weight gain of all other animals, including the slight bodyweight loss noted for one animal at 100%, were within the range seen for historic control animals.

 

The auricular lymph nodes of the vehicle control group and 25% group were considered normal in size. The nodes of the 50% and 100% groups were considered enlarged. The largest nodes were found in the highest dose group. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

 

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, 6.8 and 10.2, 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 29.0 % was calculated.

 

Table 1. Results from the definitive test

group

TS #1

animal

Size nodes #2

DPM #3/ animal

mean

mean

(%)

left

right

DPM ± SEM #4

SI ± SEM

1

0

1

n

n

546

403

± 67

1.0

± 0.2

2

n

n

454

3

n

n

229

4

n

n

#5

5

n

n

383

2

25

6

n

n

920

751

± 74

1.9

± 0.4

7

n

n

591

8

n

n

555

9

n

n

844

10

n

n

847

3

50

11

+

+

2055

2746

± 228

6.8

± 1.3

12

+

+

2350

13

+

+

3129

14

+

+

3170

15

+

+

3028

4

100

16

+

+

1984

4101

± 628

10.2

± 2.3

17

+

+

3877

18

++

++

5599

19

+

+

3904

20

+

+

5142

# 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

# 5. Value (174) rejected and not used for interpretation since animal showed rales and bodyweight loss. Based on the available data, it was considered that the study outcome was not adversely affected.

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 29%.
Executive summary:

The study was performed to GLP and the method followed OECD 429 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 undiluted and 50% v/v in acetone/olive oil 4:1 diluted 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% 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. Very slight erythema was scored for the animals in the 100% group on Days 1, 2 and 3 post-dosing however no signs of irritation were observed thereafter. 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 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)). No irritation of the ears was observed in any of the animals examined. Body weights were within the range seen for historic control animals. The auricular lymph nodes of the vehicle control group and 25% group were considered normal in size. The nodes of the 50% and 100% groups were considered enlarged. The largest nodes were found in the highest dose group. No macroscopic abnormalities of the surrounding area were noted for any of the animals. Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 751, 2746 and 4101 DPM, respectively. The mean DPM/animal value for the vehicle control group was 403 DPM. The SI values calculated for the substance concentrations 25, 50 and 100% were 1.9, 6.8 and 10.2, respectively. The data showed a dose-response and an EC3 value of 29.0 % was calculated. Under the conditions of this study, the test substance would be considered to be classified as skin sensitizer (category 1B) under Regulation (EC) No 1272/2008.