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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:
weight of evidence
Study period:
2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented, peer reviewed study forming part of the ICCVAM/NICEATM method validation of LLNA methods

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1999

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 442A (Skin Sensitization: Local Lymph Node Assay: DA)
Principles of method if other than guideline:
LLNA: DA determines sensitisation potential by measuring the proliferation of lymphocytes in the auricular lymph nodes draining the site of exposure (ears). The method employs non-radioactive techniques to assess lymphocyte proliferation by measuring adenosine triphosphate (ATP) content in the lymph nodes, thus eliminating the use of tritiated thymidine or iodine-125 based measurements used by the traditional LLNA.
In the traditional LLNA, the test substance is administered on three consecutive days (days 1, 2, and 3). On day 6, tritiated thymidine or iodine-125 is administered via the tail vein and the lymph nodes are excised five hours later. A lymph node cell suspension is then prepared and tritiated thymidine or iodine-125 incorporation is determined by scintillation counting. In the LLNA: DA method the test substance is applied on days 1, 2, 3, and 7. One hour prior to each application of the test substance, 1% sodium lauryl sulfate (SLS) is applied to the dorsum of the treated ears to increase absorption of the test substance across the skin. Twenty-four to 30 hours after the last test substance application, the auricular lymph nodes are excised, a lymph node cell suspension is prepared, and the ATP content is measured by luciferin-luciferase assay.
GLP compliance:
no
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): Trimellitic anhydride

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: 11 weeks
- Weight at study initiation: 20.7 - 24.4 g
- Housing:
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 25 deg C
- Humidity (%): 40 - 70%
- Air changes (per hr): 8 - 10

IN-LIFE DATES: From: 2005-08-24 To: 2005-08-31

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0.1, 0.25 and 0.5%
No. of animals per dose:
3 - 4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Soluble

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA-DA
- Criteria used to consider a positive response: Si > 3

TREATMENT PREPARATION AND ADMINISTRATION: Dorsum of both ears treated with 1% sodium lauryl sulphate (SLS). One hour later 25 µL of 3 concentrations of test substance, positive control or vehicle alone applied to the dorsum of both ears. This treatment regime was followed on Days 1, 2, 3 and 7. On Day 8 (24-30 hours after last application) draining auricular lymph nodes were excised, weighed, and pooled. Lymph node cells were crushed and spread between two glass slides and then scraped and suspended in 1 mL phosphate buffered saline PBS). The cell suspension obtained was then diluted 1:100 in PBS and a 100 µL aliquot was used to measure ATP content by luciferin-luciferase assay.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean relative light unit (RLU) value for each experimental group was calculated, and the Stimulation Index (SI) calculated as the mean ATP content in the LNC suspension obtained from the test group relative to that in the LNC suspension obtained from the control group. The cut-off point for a positive result was SI ≥3 and any result with an SI <3 was considered negative.

Results and discussion

In vivo (LLNA)

Results
Parameter:
SI
Remarks on result:
other: Vehicle - 1.00 TMA 0.1% - 2.46 TMA 0.25% - 3.58 TMA 0.5% - 4.96 Positive control (15% HCA) - 5.67

Any other information on results incl. tables

 

Body

Lymph node weight

 

ATP luminescence (RLU)

 

Treatment

weight

Individual

Mean ± SD

SI

Individual

Mean ± SD

SI

 

(g)

(mg)

(mg)

 

 

 

 

 

22.6

3.30

 

 

3101

 

 

Vehicle

22.3

3.54

3.48 ± 0.51

1.00 ± 0.15

3253

3362 ± 736

1.00 ± 0.22

control

23.1

2.93

 

 

2687

 

 

 

22.9

4.15

 

 

4407

 

 

 

21.6

10.21

 

 

22800

 

 

Positive

24.2

9.42

9.29 ± 0.76

2.67 ± 0.22

16696

19056 ± 2636

5.67 ± 0.78

control

20.7

8.38

 

 

17973

 

 

 

21.4

9.15

 

 

18757

 

 

 

21.8

4.61

 

 

5681

 

 

TMA 0.1%

23.0

6.36

5.76 ± 0.99

1.65 ± 0.29

7841

8272 ± 2831

2.46 ± 0.84

 

24.1

6.30

 

 

11293

 

 

 

22.5

7.46

 

 

13902

 

 

TMA 0.25%

23.4

6.43

6.81 ± 0.57

1.96 ± 0.16

11270

12045 ± 1615

3.58 ± 0.48

 

23.6

6.54

 

 

10963

 

 

 

21.9

8.31

 

 

14361

 

 

TMA 0.5%

24.4

10.50

9.15 ± 1.18

2.63 ± 0.34

18976

16670 ± 2308

4.96 ± 0.69

 

21.7

8.65

 

 

16673

 

 

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Trimellitic anhydride was sensitising in the mouse Local Lymph Node Assay.
Executive summary:

Trimellitic anhydride was sensitising in the reported variant of the mouse Local Lymph Node Assay, the authors stating an EC3 of 0.20% compared with an EC3 of 0.22% obtained with the traditional LLNA.