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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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)
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)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Remarks:
CBA/CaOlaHsD
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann, 33178 Borchen, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: Obtained from controlled full barrier breeding facility (SPF).
- Age at study initiation: 8 to 9 weeks
- Weight at study initiation: ca. 20 g
- Housing: Full barrier in an air-conditioned room. Animals housed in groups of 5 in IVC cages containing saw fibre bedding.
- Diet (e.g. ad libitum): Free access to Altromin maintenance diet.
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: At least 5 days.
- Indication of any skin lesions: Assessed ahead of study. Animals with lesions were not used.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C.
- Humidity (%): 55 +/- 10%
- Air changes (per hr): At least 10
- Photoperiod (hrs dark / hrs light): artificial light (12 hours light/dark cycle)

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
A solubility test was initially performed to define the vehicle and maximum concentration possible. the maximum technically applicable concentration was found to be 25% in DMSO. The concentrations used in the preliminary test were 25% in DMSO. Based on the observations in the preliminary test, concentrations of 6.25, 12.5 and 25% in DMSO were used in the main test.
No. of animals per dose:
3 females used in the preliminary test (2 at 25% and one control) and 5 females per group in the main test.
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: 25% in DMSO maximum technically applicable concentration.
- Irritation: No signs of irritation at the application site were noted.
- Systemic toxicity: No signs of systemic toxicity were observed.
- Ear thickness measurements: No effects of treatment on ear thickness.
- Erythema scores: No signs of irritation.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: SI Index =/> 3 at one concentration.

TREATMENT PREPARATION AND ADMINISTRATION: The test substance was suspended in DMSO. The preparations were made immediately prior to dosing. DMSO was used as the vehicle and served as the negative contol.

Each mouse was treated by topical application of 25 µL of the selected solution onto the dorsal surface of each ear. Applications were performed once daily over three consecutive days.
Positive control substance(s):
other: P-Phenylenediamine (CAS No. 106-50-3)
Statistics:
No relevant EC3 value could be calculated.

Results and discussion

Positive control results:
The results of the positive control assay (from the most recent reliability check) showed that the test system was working adequately.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
0.3
Variability:
Individual values 0.2 to 0.4
Test group / Remarks:
6.25% in DMSO
Parameter:
SI
Value:
0.6
Variability:
Individual values 0.6 to 0.7
Test group / Remarks:
12.5% in DMSO
Parameter:
SI
Value:
0.3
Variability:
Individual values 0.3 to 0.4
Test group / Remarks:
25% in DMSO
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: The proliferative response of lymph node cells was expressed as the number of radioactive disintergrations per minute (DPM/Node). Before DPM/Node values were calculated, background values were subtracted.

DETAILS ON STIMULATION INDEX CALCULATION: The ratio of 3H-methyl thymidine; incorporation into lymph node cells of test group animals relative to that recorded for control group animals.

EC3 CALCULATION: The EC3 value could not be calculated as all mean SI values were > 3.

CLINICAL OBSERVATIONS: All animals survived the observation period without showing clinical signs.

BODY WEIGHTS: All animals showed expected weight development.

Any other information on results incl. tables

Radioactive determination of test substance groups.

POS

CPM

Test item

Conc. (%)

Animal No.

DPM

DPM – mean background

DPM node

Stimulation index

85

4019

Control

0

16

8158

8144.4

4072.2

1

86

2506

17

5120

5106.4

2553.2

87

1176

18

2398

2384.4

1192.2

88

841

19

1726

1712.4

856.2

89

7733*

20

15908*

Nd

Nd

MV

2135.5

MV

4350.5

4336.9

2168.5

SD

1253.1

SD

2539.2

2539.2

1269.6

49

603

Test substance

6.25

1

1216

1202.4

601.2

0.3

50

616

2

1288

1274.4

637.2

0.3

51

660

3

1353

1339.4

669.7

0.3

52

490

4

1004

990.4

495.2

0.2

53

757

5

1535

1521.4

760.7

0.4

MV

625.2

MV

1279.2

1265.6

632.8

0.3

SD

86.5

SD

173.6

173.6

86.8

0

54

1296

Test substance

12.5

6

2650

2636.4

1318.2

0.6

55

1219

7

2487

2473.4

1236.7

0.6

56

272**

8

549**

Nd

Nd

Nd

57

1477

9

2993

2979.4

1489.7

0.7

58

1501

10

3093

3079.4

1539.7

0.7

MV

1373.3

MV

2805.8

2792.2

1396.1

0.6

SD

119.2

SD

246.7

246.7

123.3

0.1

61

617

Test substance

25

11

1242

1228.4

614.2

0.3

62

867

12

1772

1758.4

879.2

0.4

63

1291*

13

2646**

Nd

Nd

Nd

64

578

14

1193

1179.4

589.7

0.3

65

748

15

1522

1508.4

754.2

0.3

MV

702.5

MV

1432.3

1418.7

709.36

0.3

SD

114

SD

232.9

232.9

116.4

0.1

97

7

Background

0

 

14

0

0

0

98

7

 

15

99

8

 

15

101

5

 

10

102

7

 

14

MV

6.8

MV

13.6

SD

1

SD

1.9

* – outlier failed Nalimov

** – Outlier failed Grubbs, Nalimov and Dixon

POS – Position in counter

CPM – Counts per minute

DPM – Disintegrations per minute

MV – mean value

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the study results, the test substance is not expected to have skin sensitising properties in accordance with the CLP regulation (1272/2008, as amended).