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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Mar - 14 Apr 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study

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)
Version / remarks:
July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Health Care Inspectorate, Ministry of Health, Welfare and Sport, Den Haag, The Netherlands
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): [trade name]
- Substance type: off-white powder with lumps
- Analytical purity: 90.7%
- Impurities (identity and concentrations): 1,5-diazabicyclo[3.2.2]nonane-3-ol 8.9%
- Expiration date of the lot/batch: 20 Dec 2014
- Storage condition of test material: at room temperature in the dark
- Other: pH: 11.0 at concentration of 10%

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J (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. 9 weeks
- Weight at study initiation: Pre-screen test: 20 - 24 g; Main test: 18 - 23 g
- Housing: during experimental phase group housing in labeled Makrolon cages (MII type, height 14 cm) with a sheet of paper
- Diet: pelleted rodent diet (SM R/M-Z from Sniff Spezialdiäten GmbH, Soest, Germany)
- Water: 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 (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 Mar 2014 To: 14 Apr 2014

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
10, 25 and 50% (w/w)
No. of animals per dose:
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. Two test substance concentrations (25% and 50%) were tested.
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. White test substance remnants on the dorsal surface of the ears of both animals treated at 50% (between Days 1 and 4) did not hamper scoring for erythema. 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 50%.

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: If the results indicate a stimulation index (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 on three consecutive days (Days 1, 2 and 3). The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test substance.
On Day 6, 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) and 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.
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 200 g for 10 minutes at 4 ºC. 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 Packard scintillation counter (2800TR).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The EC3 value was determined using linear interpolation.

Results and discussion

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 (Oct 2013) 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, 1.1 and 8.0 respectively. 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 Remark
Remarks:
The SI values calculated for the substance concentrations 10, 25 and 50% were 1.5, 2.3 and 3.9 respectively. The data showed a dose-response and an EC3 value (the estimated test substance concentration that will give a SI =3) of 35.9% was calculated.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 301, 449 and 749 DPM respectively. The mean DPM/animal value for the vehicle control group was 194 DPM.

Any other information on results incl. tables

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

group

TS [%]1

animal

Size nodes2

DPM3/ animal

mean

DPM ± SEM4

mean

SI ± SEM

left

right

1

0

1

n

n

141

194

±

22

1.0

±

0.2

2

n

n

206

3

n

n

143

4

n

n

248

5

n

n

233

2

10

6

n

n

410

301

±

38

1.5

±

0.3

7

n

n

212

8

n

n

362

9

n

n

298

10

n

n

223

3

25

11

n

n

365

449

±

52

2.3

±

0.4

12

n

n

593

13

+

+

560

14

n

n

366

15

n

n

359

4

50

16

+

+

541

749

±

72

3.9

±

0.6

17

+

+

750

18

n

n

872

19

n

n

649

20

n

n

935

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

Skin reactions: No irritation of the ears was observed in any of the animals examined. White test substance remnants on the dorsal surface of the ears of the animals treated at 50% (between Days 1 and 4) did not hamper scoring for erythema.

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 majority of auricular lymph nodes were considered normal in size, except for the nodes in one animal at 25% and two animals at 50%, which were enlarged. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Applicant's summary and conclusion

Interpretation of results:
other:
Remarks:
CLP/EU GHS Category 1B (H317) according to Regulation (EC) No 1272/2008
Conclusions:
CLP: Skin sens 1B, H317
DSD: Xi, R43