Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-07-24 to 2015-09-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
The Measurements of the cell proliferation by cell counting instead of radioactive labelling. Measurement of the ear swelling after treatment was done with Oditest S0277 on the first and last test day and mass determination of circular ear punches.
Qualifier:
equivalent or similar to
Guideline:
other: Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay. 1st
Deviations:
yes
Remarks:
The Measurements of the cell proliferation by cell counting instead of radioactive labelling. Measurement of the ear swelling after treatment was done with Oditest S0277 on the first and last test day and mass determination of circular ear punches.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay. 2nd
Deviations:
yes
Remarks:
The Measurements of the cell proliferation by cell counting instead of radioactive labelling. Measurement of the ear swelling after treatment was done with Oditest S0277 on the first and last test day and mass determination of circular ear punches.
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): L173
- Substance type: inorganic
- Physical state: solid
- Analytical purity: > 99 %
- Composition of test material, percentage of components: almost pure substance
- Expiration date of the lot/batch: does not expire
- Stability under test conditions: stable
- Storage condition of test material: 10°C - 25 °C

In vivo test system

Test animals

Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, D-97633 Sulzfeld
- Age at study initiation: 64 days
- Weight at study initiation: 28 - 37 g
- Housing: MAKROLON cages (type II)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C +/- 3 °C
- Humidity (%): 55 % +/- 15 %
- Air changes (per hr): 12 - 18
- Photoperiod (hrs dark / hrs light): 12 h dark, 12 h light

IN-LIFE DATES: From: 2015-09-04 To: 2015-09-12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25 µL L174: 10%, (w/w), 25%, (w/w), 50%, (w/w)
No. of animals per dose:
6
Details on study design:
RANGE FINDING TESTS:
A preliminary experiment was carried out in 3 animals to examine the irritating potential and handling/application of the test item in order to select the appropriate concentrations. Three concentrations of 10%, 25% and 50% suspended in acetone/olive oil (4:1 v/v) were examined.
The preliminary experiment was conducted under conditions identical to the main LLNA study, except there was no assessment of lymph node proliferation and only 1 animal per concentration was used.

MAIN STUDY
Five (5) groups of 6 female animals each were examined. At the start of the experiment the mice were weighed and assigned to each of the 5 groups by a randomisation program (block size n = 6).

Group Concentration and application volume per ear Number of animals Animal no.
1 5 µL vehicle (negative control) 6 1 - 6
2 25 µL L173 (10%, w/w) 6 7 - 12
3 25 µL L173 (25%, w/w) 6 13 - 18
4 25 µL L173 (50%, w/w) 6 19 - 24
5 5 µL of a 20% solution of 6 25 - 30
alpha-hexyl cinnamic aldehyde in
acetone / olive oil (4:1, v/v)
All animals were females.

The study was performed according to OECD 429, however not employing the use of radioactive labelling to measure cell proliferation, as the radioactive method proposed by the OECD guideline led to problems in various EU laboratories. However, the OECD guideline allows other endpoints for assessment of proliferation in form of lymph node cell counts and lymph node weights if justification and appropriate scientific support exist showing the validity of this method.

The alternative method used for the study employing the lymph node weight and lymph node cell count to assess proliferation has been established by an European interlaboratory validation exercise, as described in the two publications by Ehling et al. 2005a and 2005b.

In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item.

The experimental schedule of the assay was as follows:

-Day 1:
The weight of each animal was individually identified and recorded. In addition, ear swelling measurements were carried out at the helical edge of both ears using an Oditest micrometer.
Open application of 25 µL of the appropriate dilution of the test item, the vehicle alone or the positive control (as appropriate) were administered to the dorsum of each ear.
- Days 2 and 3:
The application procedure carried out on day 1 was repeated
- Day 4 (24 hours after the last application):
Ear swelling measurements (immediately before sacrificing the mice) were carried out at the helical edge of both ears using an Oditest micrometer.
The animals were sacrificed under ether anaesthesia. Punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and immediately weighed on an analytical balance.
Lateral pairs of auricular lymph nodes draining the ear tissue were excised, carefully separated from remaining fatty tissue and weighed on an analytical balance immediately following preparation. The lymph nodes were then stored on ice in PBS /0.5% BSA and subjected to the preparation of single cell suspensions by mechanical tissue disaggregation. The cells were counted automatically in a cell counter.
Statistics:
U-test according to Mann and Whitney (lymph node weight significance; cell count).
Linear regression analysis employing Person's correlation coefficient (possible concentration-response-relationship for the lymph node weight).
Nalimov test (determination of outliners).
Standard deviation and relative standard deviation were calculated, too.

Results and discussion

Positive control results:
Parameter stimulation indices (SI)
group 5, positive control
Lymph node cell count 2.164
Lymph node weight 1.596
Ear weight 1.110
Ear thickness, TD4 1.112

In vivo (LLNA)

Results
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Main study results: stimulation indices (SI): Parameter Group 1, Group 2, Group 3, Group 4, Group 5 neg. control 10 % 25 % 50 % pos. control Lymph node cell count 1.000 1.233 1.039 1.304 2.164 Lymph node weight 1.000 1.000 0.979 1.043 1.596 Ear weight 1.000 0.995 0.995 1.005 1.110 Ear thickness, TD4 1.000 1.009 1.034 1.034 1.112 ____ significantly different from control at p ≤ 0.01

Any other information on results incl. tables

No signs of local or systemic intolerance were recorded.

The animal body weight was not affected by the treatment.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the present test conditions, L173 at concentrations of 10%, 25% or 50% (w/w) in acetone/olive oil (4:1, v/v) did not reveal any sensitising properties in the local lymph node assay.
Executive summary:

The purpose of this study was to determine the sensitising potential of L173 in the modified local lymph node assay in mice.

The study was performed according to OECD 429, however not employing the use of radioactive labelling to measure cell proliferation, as the radioactive method proposed by the OECD guideline led to problems in various EU laboratories: such as (i) practical difficulties/complexity of the test, in particular the radiochemical steps, which sometimes resulted in loss of specimen/activity; this in turn led to variability in the results and to a poor reproducibility and (ii) radiation protection issues. However, the OECD guideline allows other endpoints for assessment of proliferation in form of lymph node cell counts and lymph node weights if justification and appropriate scientific support exist showing the validity of this method.

The alternative method used for the study employing the lymph node weight and lymph node cell count to assess proliferation has been established by an European interlaboratory validation exercise, as described in the two publications by Ehling et al. 2005a and 2005b.This method has the advantage of (i) more simplistic experimental work, (ii) less variability, (iii) better reproducibility, (iv) faster results, (v) reduced costs.

In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item. It is important to determine if a positive test result is due to the skin irritation potential of the test item or due to its sensitising properties.

Stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the vehicle treated ones.

Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the interlaboratory validation of this method (Ehling et al. 2005a and 2005b)).

Three concentrations of L173 (10%, 25% and 50% (w/w), suspended in acetone/olive oil (4:1, v/v))(w/w) were tested in six female NMRI mice per group and compared to a vehicle control group.

A 50% suspension was the highest feasible concentration of L173 in acetone/olive oil (4:1, v/v).

In addition, a positive control group (20% solution v/v of alpha-hexyl cinnamic aldehyde in acetone/olive oil (4:1, v/v)) was employed.

Stimulation indices (SI):

Parameter

Group 1, negative control

Group 2,

10%

Group 3, 25%

Group 4, 50%

Group 5,

positive control

Lymph node cell count

1.000

1.233

1.039

1.304

2.164

Lymph node weight

1.000

1.000

0.979

1.043

1.596

Ear weight

1.000

0.995

0.995

1.005

1.110

Ear thickness, TD4

1.000

1.009

1.034

1.034

1.112

____ significantly different from control at p ≤ 0.01

In the main study treatment with L173 at concentrations of 10%, 25% or 50% did not reveal any statistical significantly increased values for the lymph node cell count. The stimulation indices of the lymph node cell count did not exceed the threshold level of 1.4. In addition, no significant increase in the lymph node weight was observed. Hence, the test item is classified as not sensitising.

The threshold level for the ear weight of 1.1 was not exceeded and no increase of ear thickness was observed, i.e. no irritating properties were noted.

The positive control group caused the expected increases in lymph node cell count and lymph node weight (statistically significant at p ≤ 0.01).Therefore, the study can be regarded as valid.

No signs of local or systemic intolerance were recorded. The animal body weight was not affected by the treatment.

In conclusion, under the present test conditions, L173 at concentrations of 10%, 25% or 50% (w/w) in acetone/olive oil (4:1, v/v) did not reveal any sensitising properties in the local lymph node assay.