Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted in 2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Landesinstitut für Arbeitsschutz und Produktsicherheit, München, Germany)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsD
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann, Borchen, Germany
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: 19 - 22 g
- Housing: The animals were housed in groups of 3 (pre-screen test) and 5 (main study) in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding.
- Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water: tap water, sulphur acidified to a pH value of approximately 2.8, ad libitum
- Acclimation period: at least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
12.5, 25 and 50% (w/v)
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The maximum technically applicable concentration of the test substance was found to be 50% in acetone/olive oil (4:1) (AOO).
- Irritation: Two animals were treated with 50% test substance in AOO. A third animals was treated with vehicle only and served as negative control. No signs of systemic toxicity or skin irritation (ear swelling, erythema and eschar formation) were detected in the test substance-treated animals. However, alopecia was observed at the apllication site.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: ³H-Methylthymidine incorporation determined by β-scintillation.
- Criteria used to consider a positive response: The substance is regarded as skin sensitiser if at least one concentration of the test substance results in a 3-fold or greater increase in ³H-Methylthymidine incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of the control group (SI ≥ 3.0).

TREATMENT PREPARATION AND ADMINISTRATION: Each mouse was treated by topical application of 25 µL of a test substance solution or the vehicle alone to the dorsal surface of each ear. Topical applications were performed once daily over three consecutive days.
Five days after the first topical application, all mice were dosed with 20 µCi ³H-Methylthymidine by intravenous injection of 250 µL ³H-Methylthymidine into the tail vein. Approximately 5 hours after the injection, all mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). The cell suspension was washed with PBS, pelleted and resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. The ³H-Methylthymidine incorporation was measured in a β-counter and expressed as the number of disintegrations per minute (DPM) individually for each animal.
Positive control substance(s):
other: 1% p-phenylenediamine (reliability check)
Statistics:
Outlier tests for statistical significance were performed according to Grubbs, Nalimov and Dixon.

Results and discussion

Positive control results:
A reliability test with the positive control substance was routinely performed 2 months prior to the present study. A mean SI value of 11.3 ± 1.7 (n = 5) was determined.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 12.5% test substance: 1.4 25% test substance: 1.5 50% test substance: 1.9 negative control: 1.0
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 12.5% test substance: 1902.4 25% test substance: 2022.8 50% test substance: 2505.8 negative control: 1346.2

Any other information on results incl. tables

All animals survived until the end of the study period without showing any clinical signs or signs of dermal irritation at the application site. Alopecia was observed at the application site from Day 3 until the end of the study period in all animals treated with the test substance at a concentration of 50%. The treatment with the test substance did not influence the body weight gain of the treated animals.

Table 1. Radioactive determination of ³H-Methylthymidine incorporation

 

Animal No.

CPM

DPM

DPM - background

SI

12.5% test substance

1

789

1626

1573.8

1.2

2

1107

2283

2230.8

1.7

3

827

1721

1668.8

1.3

4

1102

2294

2241.8

1.7

5

772

1588

1535.8

1.2

mean

919.4

1902.4

1850.2

1.4

SD

152.2

318.2

318.2

0.2

25% test substance

6

1033

2145

2092.8

1.6

7

837

1738

1685.8

1.3

8

2705*

5643*

---

---

9

1241

2538

2485.8

1.9

10

812

1670

1617.8

1.3

mean

980.8

2022.8

1970.6

1.5

SD

172.9

348.5

348.5

0.3

50% test substance

11

1015

2096

2043.8

1.6

12

1298

2679

2626.8

2.0

13

1275

2686

2633.8

2.0

14

1820**

3777**

---

---

15

1241

2562

2509.8

1.9

mean

1207.3

2505.8

2453.6

1.9

SD

112.8

241.6

241.6

0.2

negative control

16

243

497

444.8

---

17

479

988

935.8

---

18

667

1375

1322.8

---

19

981

2014

1961.8

---

20

903

1857

1804.8

---

mean

654.6

1346.2

1294.0

1.0

SD

271.5

558.1

558.1

---

Background value

mean

25.8

52.2

---

---

SD

1.5

3.5

---

---

* outlier, failed Grubbs, Nalimov and Dixon

** outlier, failed Nalimov and Dixon

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
CLP: not classified
DSD: not classified