Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 June 2010 - 23 June 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material : J-37
- Substance type: red powder
- Physical state: powder
- Analytical purity: 99.3%
- Lot/batch No.: 91116
- Expiration date of the lot/batch: 16th April 2011
- Storage condition of test material: at room temperature in the dark in desiccators
- Stability under test conditions: stable

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approximately 9 weeks old
- Weight at study initiation: body weight variation was within ± 20% of the sex mean.
- Housing: individual housing in labeled Macrolon cages (MI type; height 12.5 cm) containing sterlised sawdust as bedding material (Litalabo, S. P. P. S., Argenteuil, France). Paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Lts), Surrey, UK) was supplied as cage-enrichment. The paper was removed on Day 1 prior to dosing and was supplied again after scoring of the ears on Day 3.
- Diet: free access to pelleted rodent diet (SM R/M from SSNIFF Spezialdiaten GmBH, Soest, Germany).
- Water: free access to tap water.
- Acclimation period: 5 days before start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0 °C (acutal range: 20.8 - 23.1)
- Humidity (%): 40 - 70 (actual range: 41 - 76%)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours artifical fluorescent light and 12 hours darkness per day.

IN-LIFE DATES: From:07 June 2010 To: 23 June 2010

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 5, 10 and 25%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Irritation: no irritation and no signs of systemic toxicity were observed in any of the animals examined. Purple remnants of the test substance prevented scoring of erythema of both ears of both animals, but no oedema was observed. Based on these results, the highest test substance concentration selected for the main study was 25%.

MAIN STUDY
- Criteria used to consider a positive response: If the result indicates a SI ≥3, the test substance may be regarded as a skin sensitiser, based on the test guideline and recommendations done by ICCVAM.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The SI values calculated for the substance concentrations 5, 10 and 25% were 1.7, 2.7 and 8.8 respectively. An EC3 of 10.7% 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 months HCA reliability checks in CBA/J female mice of the recent years were 14.1, 13.8, 13.9, 16.0, 11.9 and 16.9%. Based on the results, it was concluded that the Local Lymph Node Assay in the mouse as supplied by Janvier performed at NOTOX is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The SI values calculated for the substance concentrations 5, 10 and 25% were 0.8, 1.4 and 0.7 respectively.
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 417, 713 and 361 DPM respectively. The mean DPM/animal value for the vehicle control group was 509 DPM

Any other information on results incl. tables

Skin reactions/ irritation:

No irritation of the ears was observed in any of the animals examined. No oedema was observed in any of the animals examined. Purple staining by the test substance prevented scoring for erythema. 

Body weight:

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 in some animals, was considered not toxicologically significant.

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Table 2 : Skin reactions, body weights and relative size auricular lymph nodes

Group

Test substance1(% w/w)

An2

Day1

Day 3

Skin reactions dorsal surface ear

Day 6

Bw (g)3

Left

Right

Bw (g)3

Size Nodes4

erythema

oedema

erythema

oedema

Left

Right

1

0% (Vehicle)

1

22

0

0

0

0

23

n

n

2

22

0

0

0

0

22

n

n

3

25

0

0

0

0

24

n

n

4

22

0

0

0

0

22

n

n

5

22

0

0

0

0

22

n

n

2

5%

6

21

6

0

6

0

21

n

n

7

24

6

0

6

0

23

n

n

8

23

6

0

6

0

22

n

n

9

23

6

0

6

0

23

n

n

10

24

6

0

6

0

22

n

n

3

10%

11

22

6

0

6

0

22

n

n

12

21

6

0

6

0

21

n

n

13

23

6

0

6

0

23

n

n

14

24

6

0

6

0

23

n

n

15

23

6

0

6

0

22

n

n

4

25%5,6

16

22

6

0

6

0

22

n

n

17

23

6

0

6

0

22

n

n

18

21

6

0

6

0

22

n

n

19

22

6

0

6

0

22

n

n

20

24

6

0

6

0

23

n

n

1.      Vehicle: Acetone/olive oil (4:1).

2.      Animal number

3.      Body weight (grams)

4.      Relative size auricular lymph nodes( n: considered to be normal)

5.      Applied using a pipette with the tip cut off.

6.      No scoring due to purple staining of the ear. 

Table 3. Disintegrations Per Minute (DPM) and Stimulation Index (SI).

Group

Test substance1(% w/w)

Mean

DPM ± SEM

SI ± SEM

2

5%

417 ± 64

0.8 ± 0.2

3

10%

719± 180

1.4 ± 0.4

4

255

361 ± 61

0.7 ± 0.2

1

0% (Vehicle)

509 ± 83

1.0 ± 0.2

1.      Vehicle: Acetone/olive oil (4:1).

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information