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
Study period:
2015-10-07 to 2015-11-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant study report.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(22 July 2010)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(06 July 2012)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Envigo CRS GmbH, In den Leppsteinswiesen 19, 64380 Rossdorf, Germany
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Substance type: organic
- Physical state: metallic green-violet solid, melt (highly viscous)
- Analytical purity: 91.6 %
- Expiration date of the lot/batch: 23 January 2019
- Storage condition of test material: at room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Postbus 6174, 5960 AD Horst, The Netherlands
- Age at study initiation: 9 - 12 wks (pre tests); 10 - 11 wks (main study)
- Weight at study initiation: 18 - 23.4 g
- Housing: group wise
- Diet: ad libitum, 2018C Teklad Global 18 % protein rodent diet
- Water: ad libitum, tap water
- Acclimation period: at least five days prior to start of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45 - 65
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
0.1, 0.5, 2 %
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was a 50 % suspension in DMF. Grinding of the test item in a mortar was used to formulate the test item.
To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, three pre-tests were performed in two animals each. The two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 25 and 50 % (pretest 1), 5 and 10% (pre test 2) and 1 and 2 % (pre test 3) once daily each on three consecutive days. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm²) and were immediately pooled per animal and weighed using an analytical balance.

MAIN STUDY
Topical application
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (d = 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).

Administration of 3H-methyl-thymidine
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 20.3 µCi of 3H-methyl thymidine (equivalent to 81.3 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Determination of incorporated 3HTdR
Approximately five hours after treatment with 3HTdR all mice were euthanised by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death. The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a beta-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute.

Determination of Lymph Node Weight and Cell Count
After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance. Furthermore, the lymph node cell count was determined for each animal. For this, the volume of the cell suspensions was adjusted to an equal final volume and vortexed. Subsequently, individual cell counts were determined using a cell counter (CASY® DT, Schärfe System). The values obtained were recorded manually.

Determination of Ear Weights
After the lymph nodes had been excised, both ears (left and right) of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm²). For each animal both punches were immediately weighed (pooled per animal) using an analytical balance.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers (performed with validated program R Script Outlier.Rnw).

Results and discussion

Positive control results:
The periodic positive control experiment was performed using CBA/CaOlaHsd mice in October 2015. The reliability check with alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at Envigo CRS is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: In this study, Stimulation Indices (S.I.) of 1.05, 4.14, and 7.62 were determined with the test item at concentrations of 0.1, 0.5, and 2 % (w/w) in DMF, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations of 0.1, 0.5 and 2 % were 1107.2, 4382.4 and 8072.6 DPM , respectively. The mean DPM/animal value for the vehicle control group was 1059.4 DPM.

Any other information on results incl. tables

Pre Tests

At the tested concentrations of the first pre-test, the animals did not show any signs of systemic toxicity. Redness of the ear skin could not be observed due to the colour of the test item. On day 6, the animal treated with 50 % test item concentration showed eschar formation of the ear skin (per grading table defined as score 4) as well as a swollen face. Both animals showed stiffened ears from day 2 onwards and an increased ear weight above the threshold recommended by OECD.

In the second pre-test, the animals did not show any signs of systemic toxicity at 5 and 10 %. Redness of the ear skin could not be observed due to the colour of the test item. On day 6, upon preparation, both animals showed eschar formation of the ear skin (defined as score 4). Due to the eschar formation a third pre-test was conducted.

At the tested concentrations of 1 and 2 % the animals did not show any signs of local irritation or systemic toxicity. Redness of the ear skin could not be observed due to the colour of the test item.

Main Test

No animal died during the course of the study. No systemic signs of toxicity were observed. Redness of the ear could not be assessed due to the colour of the test item. The body weight of the animals, recorded prior to the first application and prior to treatment with ³HTdR, was within the range commonly recorded for animals of this strain and age. The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant and biologically relevant increase was observed in lymph node weight and -cell count in the mid and high dose group in comparison to the vehicle control group. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was exceeded in the mid and high dose group.

The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A biologically relevant or statistically significant increase in ear weights was not observed. Furthermore, the cut-off value (1.1) of the ear weight index for a positive response regarding ear skin irritation reported for BALB/c mice was not reached or exceeded in any of the treated groups.

 

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information