Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2013 to March 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to OECD guideline under GLP
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Principles of method if other than guideline:
Not relevant
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

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

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
Number of animals for
the pre-test: 2 females
Number of animals for
the main study: 24 females
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) groups: 2 (1 group for the test item, 1 group for positive control)
Number of positive control groups: 1
Age: Pre-test: 10 - 11 weeks (beginning of treatment)
Main study: 8 - 9 weeks (beginning of treatment)
Body weight: see Appendix 1 and 2
Identification: The animals were distributed into the test groups at random. All animals belonging to the same experimental group were kept in one cage. In the main experiment, the animals were identified by tail tags. In the pre-experiment, animals were identified by cage number.
Acclimation: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
The highest test item concentration, which could be technically used was a 10% suspension in PG.
No. of animals per dose:
4 females (nulliparous and non-pregnant)
Details on study design:
Topical application
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 2.5, 5 and 10% in PG. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (  8 mm) of each ear once daily for three consecutive days. Two further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).

Determination of Ear Thickness
Prior to the first and third application of the test item (study days 1 and 3) and prior to treatment with 3HTdR (study day 6), the ear thickness was determined using a micrometer.


Administration of 3H-methyl-thymidine
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 19.6 µCi of 3H-methyl thymidine (equivalent to 78.4 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 for each experimental group (8 nodes per group). 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 -scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The -scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute.

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.

Interpretation of raw data
The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/lymph node) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/lymph node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
• First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
• Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables.
Where appropriate, the EC3 value was calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.
The ANOVA (Dunnett-test) was conducted on the ear weights to assess whether the difference is statistically significant between test item groups and negative control (vehicle) group.
Also, a statistical analysis was conducted on the ear thickness values to assess whether a statistically significant increase in ear thickness could be observed when comparing the values measured on day 1 prior to application with the values measured on days 3 or 6 in the respective test item groups or within the vehicle control group.
For all statistical calculations SigmaStat for Windows (Version 2.0) was used.
However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:
The sensitivity and reliability of the experimental technique employed was assessed by use of α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1, v/v) (compound listed in OECD 429 Guideline) which is known to have skin sensitisation properties in mice. The periodic positive control experiment was performed using CBA/CaOlaHsd mice in April 2013.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: In this study Stimulation Indices (S.I.) of 1.66, 1.39 and 1.72 were determined with the test item at concentrations of 2.5, 5 and 10% formulated in PG, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Concentration % Group Measurement DPM Calculation Result DPM-BGa) number of lymph nodes DPM per lymph nodeb) S.I. --- BG I 19 --- --- --- --- --- BG II 45 --- --- --- --- 0 1 2604 2572.0 8 321.5 1.00 2.5 2 4292 4260.0 8 532.5 1.66 5 3 3603 3571.0 8 446.4 1.39 10 4 4450 4418.0 8 552.3 1.72 0 5 6322 6290.0 8 786.3 1.00 25 6 34535 34503.0 8 4312.9 5.49

Any other information on results incl. tables

Calculation and Results of Individual Data

Vehicle: PG

 

Concentration %

Group

Measurement DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

19

---

---

---

---

---

BG II

45

---

---

---

---

0

1

2604

2572.0

8

321.5

1.00

2.5

2

4292

4260.0

8

532.5

1.66

5

3

3603

3571.0

8

446.4

1.39

10

4

4450

4418.0

8

552.3

1.72

0

5

6322

6290.0

8

786.3

1.00

25

6

34535

34503.0

8

4312.9

5.49

1    =  Control Group (PG)

2-4=  Test Groups (K-36)

5    =  Control Group for Positive Control (acetone/olive oil (4+1, v/v))

6    =  Positive Control Group (Alpha-Hexylcinnamaldehyde; HCA)

a)   =  The mean value was taken from the figures BG I and BG II

b)    =  Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Migrated information
Conclusions:
The test item K-36 was not a skin sensitiser under the test conditions of this study.
Executive summary:

In the study the test item K-36 formulated in PG was assessed for its possible skin sensitising

potential.

For this purpose a local lymph node assay was performed using test item concentrations of 2.5, 5

and 10%. The highest concentration tested was the highest concentration that could be technically

achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the

pre-experiment.

The animals did not show any signs of systemic toxicity during the course of the study and no

cases of mortality were observed. The observation of skin irritation was not possible due to

inherent color of the test item.

On day 3 and 4, the animals treated with positive control substance showed an erythema of the

ear skin (Score 1).

In the groups treated with a test item concentration of 2.5 and 10%, a statistically significant

increase (p < 0.05) in ear weight was observed in comparison to the values of the vehicle control

group. However, this increase was considered to be not biologically relevant.

In this study Stimulation Indices (S.I.) of 1.66, 1.39 and 1.72 were determined with the test item

at concentrations of 2.5, 5 and 10% formulated in PG, respectively.

The test item K-36 was not a skin sensitiser under the test conditions of this study.