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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 - 29 Sep 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/J strain, inbred, SPF-Quality
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approx. 10 weeks old
- Weight at study initiation: Pre-screen test: 20-24 g, Main test: 19-25 g
- Housing: group housed in Makrolon cages (MIII type; height 18 cm), sterilized sawdust was used as bedding material, paper and shelters as cage-enrichment
- Diet: pelleted rodent diet, SM R/M-Z (SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

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

IN-LIFE DATES: From: 10 Sep 2014 To: 29 Sep 2014
Vehicle:
methyl ethyl ketone
Concentration:
5, 10 and 25% (w/w)
No. of animals per dose:
range finding study: 2 females
main study: 5 females
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 (10% and 25%) were tested. 25% was the highest concentration that could be prepared homogeneously.
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 prior to dosing on Days 1 and 3, and on Day 6.
No irritation and 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. White staining of the dorsal surface of the ears by test substance remnants was noted for all animals (10% Days 1-3; 25% Days 1-4). Based on these results, the highest test substance concentration selected for the main study was 25%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by ß-scintillation
- Criteria used to consider a positive response: A Stimulation Index (SI) was calculated for each group using the individual SI values. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION: 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 application was repeated on Days 2 and 3; local irritation reactions were assessed. On Day 6 an injection of 250 µl phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine (3H-TdR) was made into the tail vein of each experimental mouse. Five hours later, following excision of the nodes, 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 and stored in the refrigerator until the next day.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The results of a reliability test with three concentrations of hexyl cinnamic aldehyde in acetone/olive oil (4:1 v/v) was performed not more than 6 months previously (Apr 2014) using the same materials, animal supplier, animal strain and essential procedures. The SI values calculated for the substance concentrations 5, 10 and 25% were 1.2, 1.4 and 4.7 respectively. An EC3 value of 17.3% 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 16.9, 14.4, 16.5, 14.5, 13.4 and 14.1%.
Parameter:
SI
Remarks on result:
other: The SI values calculated for the substance concentrations 5, 10 and 25% were 1.2, 1.2 and 1.3 respectively. It was established that the EC3 value (the estimated test substance concentration that will give a SI =3) (if any) exceeds 25%.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 10 and 25% were 788, 843 and 865 DPM respectively. The mean DPM/animal value for the vehicle control group was 684 DPM.

Table1: Relative size lymph nodes, radioactivity counts (DPM) and Stimulation Index (SI).

Group

TS1(%)

Animal

Size nodes2

DPM3/aninmal

Mean
DPM
±SEM4

Mean
SI
±SEM

left

right

1

0

1

n

n

863

684 ± 129

1.0 ± 0.3

2

n

n

191

3

n

n

915

4

n

n

764

5

n

n

688

2

5

6

n

n

1117

788 ± 98

1.2 ± 0.3

7

n

n

725

8

n

n

782

9

n

n

504

10

n

n

810

3

10

11

n

n

521

843 ± 123

1.2 ± 0.3

12

n

n

1144

13

n

n

1090

14

n

n

838

15

n

n

621

4

25

16

n

n

1128

865 ± 93

1.3 ± 0.3

17

n

n

722

18

n

n

749

19

n

n

860

205

n

 

 

1TS = test substance (% w/w)

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

3DPM= Disintegrations per minute

4SEM = Standard Error of the Mean

5Right node was not found during section. The DPM value for this animal was not used for interpretation since this value is based on one node only. Based on the available data, it was considered that the study outcome was not adversely affected.

Skin reactions: No irritation of the ears was observed in any of the animals examined. White staining of the dorsal surface of the ears by test substance remnants was noted for all experimental animals between Days 1 and 3. The staining did not hamper the scoring of the ears.

Systemic toxicity and body weights: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Macroscopy of the auricular lymph nodes and surrounding area: The auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
CLP: not classified
DSD: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The skin sensitising potential of the test substance was investigated in a Local Lymph Node Assay (LLNA) in mice according to OECD Guideline 429 and in compliance with GLP (Latour, 2014). Based on the results of a pre-screen test, test substance concentrations of 5%, 10% and 25% (w/w) in methyl ethyl ketone were selected for the treatment of mice in the main study. In this experiment, 5 female CBA/J mice per test group were treated with the test substance or vehicle methyl ethyl ketone alone for three consecutive days by open application on the ears (25 µL/ear). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 10 and 25% were 788, 843 and 865 DPM respectively. The mean DPM/animal value for the vehicle control group was 684 DPM. The SI values calculated for the substance concentrations 5, 10 and 25% were 1.2, 1.2 and 1.3 respectively. It was established that the EC3 value (if any) exceeds 25%. No irritation of the ears was observed in any of the animals examined. The auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted in any of the animals. The six-month reliability check with hexyl cinnamic aldehyde indicated that the LLNA is an appropriate model for testing for contact hypersensitivity. Based on the results, the test substance was not regarded as a skin sensitizer under the conditions of the test.


Migrated from Short description of key information:
skin sensitisation (OECD 429): not sensitising

Justification for selection of skin sensitisation endpoint:
The reliable GLP compliant OECD Guideline study was chosen.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:
Justification for selection of respiratory sensitisation endpoint:
Study not required according to Annex VII-X of Regulation (EC) No 1907/2006.

Justification for classification or non-classification

The available data on sensitisation of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.