Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

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:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS GmbH, C/O Postfach 553 NL-5800 AN Venray
- Age at study initiation: 8 weeks (main test); 9 weeks (pretest)
- Weight at study initiation: 17.2 g- 20.1 g (main test), 20.1 g - 22.9 g (pretest)
- Housing: single (Polycarbonate cages type MII)
- Diet (e.g. ad libitum):Kliba mouse/rat maintenance diet "GLP" supplied by Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum.
- Water (e.g. ad libitum): drinking water ad libitum
- Acclimation period: 5 days before the first application

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 45 - 65%
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
methyl ethyl ketone
Concentration:
2.5, 10 and 25% (w/w)
No. of animals per dose:
5
Details on study design:
The study comprised three treatment groups and a vehicle control group. Each group consisted of 5 mice. Prior to first application, the animals were distributed to the individual groups, received animal numbers and were allocated to the respective cages according to the randomization instructions. Individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.
Obvious signs of systemic toxicity and/or local inflammation atthe application sites were noted for each animal in the raw data. A check for moribund and dead animals was made twice each workday (beginning and end) and once on Saturdays, Sundays
and on public holidays.
Epicutaneous application is simulating dermal contact with the compound, which can occur under practical use conditions. Application volume of 25 µL per ear. Site of application dorsal part of both ears. Frequency of application 3 consecutive applications (day 0 – day 2) to the same application site.
The animals of control group 1 and test groups 2-4 were treated with the vehicle or a test-substance preparation as given in section 2.2.
On study day five (about 66 to 72 hours after the last application of test substance to the ears), 20 μCi 3H-thymidine in 250 μL sterile saline were injected into the tail vein of the mice.
The animals were sacrificed on study day 5 about 5 hours after 3H-thymidine injection by cervical dislocation under Isoflurane anesthesia.
Immediately after the death of each animal, a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each animal. These measurements serve for detecting a potential inflammatory ear swelling.
Immediately after removal of the ear punches, the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal.
After weight determination, the pooled lymph nodes of each animal were stored in phosphate-buffered saline (PBS) in an ice water bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully passing the lymph nodes through an iron mesh (mesh size 200 μm) into 6 mL phosphate-buffered physiological saline. For determination of cell counts, an aliquot of each suspension was further diluted with Casy®ton in a ratio 1:500. The cell count was determined using a Casy® Counter.
The remaining cell suspensions were washed twice with PBS and precipitated with 5% trichloro-acetic acid (TCA). Each precipitate was transferred to scintillation fluid and incorporation of 3H-thymidine into the cells was measured in a ß-scintillation counter.
Positive control substance(s):
other: A positive control with a substance that has been proven to cause skin sensitization in the animal strain used will not be included in this study. A separate study with a positive control is carried out in the laboratory twice a year.
Statistics:
Cell count, 3H-thymidine incorporation, lymph node weight and ear weight, WILCOXON - Test

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
EC3
Remarks:
%
Value:
9.1
Parameter:
other: EC1,5
Remarks:
%
Value:
8.6

Any other information on results incl. tables

Test item concentration

Group Calculation

Mean DPM per animal (2 lymph nodes)

SD

S.I.

Vehicle Control Group (vehicle: MEK)

304.9

125.3

1.00

2.5% C9-11 Methacrylat

 

350.4

108.2

1.15

10%C9-11 Methacrylat

 

995.7

207.6

3.27

20% C9-11 Methacrylat

 

2144.8

611.8

7.03

Stimulation indices:

Test Group

Treatment

3H-thymidine incorporation Stimulation Index1

Cell Count Stimulation Index1

Lymph Node Weight

Stimulation Index1

Ear Weight Stimulation Index1

1

Vehicle MEK

1.00

1.00

1.00

1.00

2

2.5% in MEK

1.15

0.92

0.92

1.01

3

10% in MEK

      3.27 ##

    1.63 ##

    1.56 ##

   1.16 #

4

25% in MEK

     7.03 ##

    2.28 ##

    2.17 ##

     1.70 ##

1SI-calculation versus vehicle control

The statistical evaluations were performed using the WILCOXON-test (# for p ≤ 0.005, ## for p ≤ 0.01)

The threshold concentration for sensitization induction was >2.5% <10%. The EC 3 (estimated concentration that leads to the SI of 3.0) for 3H-thymidine incorporation and the EC 1.5 (estimated concentration that leads to the SI of 1.5) for cell count was calculated by linear regression from the results of these concentrations to be 9.1% and 8.6%, respectively.

No relevant influence on the body weights was observed during the study.

No signs of systemic toxicity were noticed in all animals during general observation.

Slight scaling at the ear root was observed on four animals at the 10% concentration on study day 5. A very slight erythema was observed on all animals at the 25% concentration on study days 1 and 2. Slight scaling and a slight incrustation were observed on all animals at the 25% concentration on study day 2. On study day 5, the scaling and incrustation on all animals were moderate.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Thus, it is concluded that C9-11 Methacrylate exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.