[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 July 2017 to 23 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

yes

Remarks:

minor variation in animal room temperature with no impact on results or integrity of the study (see below)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

yes

Remarks:

minor variation in animal room temperature with no impact on results or integrity of the study (see below)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Test material form:

liquid: viscous

Details on test material:

- Appearance/physical state: Brown viscous liquid
- Storage conditions: Room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

ANIMAL INFORMATION
- Test system: Mice, CBA/CaOlaHsd (recognised as the recommended test system).
- Source: Envigo RMS B.V. Inc, Postbus 6174, 5960 AD Horst, The Netherlands.
- Number of animals for the pre-test: 2 females.
- Number of animals for the main study: 30 females.
- Number of animals per group: 5 females (nulliparous and non-pregnant).
- Number of test groups: 3
- Number of control (vehicle) groups: 2
- Number of positive control groups: 1
- Age (beginning of treatment): Pre-test: 10 to 11 weeks; Main study: 9 to 10 weeks
- Body weight: see Appendices 1 and 3 (attached).
- 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.

ANIMAL HUSBANDRY
- Housing: group.
- Cage Type: Makrolon Type II (pre-test) / III (main study), with wire mesh top.
- Bedding: granulated soft wood bedding.
- Feed: 2018C Teklad Global 18 % protein rodent diet (certified), ad libitum.
- Water: tap water, ad libitum.
- Environment: temperature 22  2 °C; relative humidity approximately 45 to 65 % (except for deviations); artificial light 6.00 a.m. to 6.00 p.m.

ANIMAL ALLOCATION
- The animals were distributed to the different test groups as shown in the table below.

Study design: in vivo (LLNA)

Vehicle:

other: cottonseed oil

Remarks:

water free; highly refined; low acidity

Concentration:

Test item concentrations of 2.5, 5, and 10 %.

No. of animals per dose:

Five

Details on study design:

PURPOSE OF TEST
- The test is designed to assess the skin sensitisation potential (delayed type hypersensitivity) of the test item in the mouse following topical application to the dorsal surface of the ear. The basic principle underlying the Local Lymph Node Assay (LLNA) is that sensitisers induce a primary proliferation of lymphocytes in the lymph node draining the application site. Primary lymphocyte proliferation is assessed during the sensitising (induction) phase of the response. This proliferation is proportional to the dose applied (and to the potency of the allergen) and provides a simple means of obtaining an objective, quantitative measurement of sensitisation.
- The LLNA assesses this proliferation as a dose response in which the proliferation in test groups is compared to that in vehicle treated controls. Lymphocyte proliferation is quantified by measuring the incorporation of radiolabelled thymidine into lymph node cells by β-Scintillation Counting. This study should provide a rational basis for risk assessment to the sensitising potential of the test item in man.

CHEMICALS
- 3H-Methyl thymidine (Perkin Elmer; aqueous solution; specific activity: 74 GBq/mmol (2 Ci/mmol); concentration: 37 MBq/mL (1 mCi/mL.
- Trichloroacetic acid (minimum purity 99.5 %)
- Phosphate buffered saline: one tablet dissolved in 200 mL deionised water

POSITIVE CONTROL ITEM
- The positive control item, Alpha-Hexylcinnamaldehyde, tech. 85%, was weighed into a volumetric flask on a tared balance and acetone:olive oil (4+1 v/v) was quantitatively added to achieve a concentration of 25% (w/v).
- The preparations were freshly made before each dosing occasion.

VEHICLE AND DOSE SELECTION
- 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 25 % suspension in cotton seed oil. Vortexing, sonicating, and warming to 37 °C were used to formulate the test item. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. vortexing, sonicating, warming to 37°C).
- To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals and stated in raw data and report. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10 and 25 % once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. 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. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥ 3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6 (for detailed results see Appendix 1).
- At the tested concentrations the animals did not show any signs of systemic toxicity. From day 2 to 6, the animals showed an erythema of the ear skin (Score 1 to 3, see Appendix 1 for details) and scaly skin on head on day 6. The animal treated with 25% test item concentration showed scaly ears on days 4 and 5 and visible swelling of ears on day 6, as well as a gain in ear thickness above the threshold (gain 62 %) and an erythema score of 3. Thus, the test item in the main study was assayed at 2.5, 5, and 10 %. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

TEST ITEM PREPARATION
- The test item was placed into an appropriate container on a tared balance and cotton seed oil was added.
- The different test item concentrations were prepared individually. Homogeneity of the test item in vehicle was maintained during treatment using a magnetic stirrer.
- The preparations were made freshly before each dosing occasion.

TEST ITEM ADMINISTRATION
- 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 cotton seed oil.
- 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.
- Three further groups of mice (two vehicle control groups and one positive control group) were treated with an equivalent volume of the relevant vehicle alone or with the positive control item at 25% (w/v).

ADMINISTRATION OF 3H-METHYL THYMIDINE
- Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 20.5 μCi of 3H-methyl thymidine (equivalent to 82 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

TERMINAL PROCEDURE
- 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.

PREPARATION OF SINGLE CELL SUSPENSIONS
- 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 twice 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 approximately 18 hours for precipitation of macromolecules.

DETERMINATION OF CELLULAR PROLIFERATION (INCORPORATION OF 3HTdR)
- 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 β-scintillation counter.
- Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid.
- The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute.

CLINICAL OBSERVATIONS
- All animals were observed on a daily basis, including pre- and post-dose observations on days 1, 2 and 3.
- Any clinical signs of systemic toxicity, local skin irritation or signs of ill health during the study were recorded.

DETERMINATION OF EAR THICKNESS
- In the pre-test, the ear thickness was determined prior to the first application of the test item (day 1), on day 3, and on day 6 prior to sacrifice using a micrometer. In the main experiment the ear thickness was determined daily.

DETERMINATION OF EAR WEIGHTS
- In the pre-test, after the lymph nodes have been excised, both ears 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 per animal using an analytical balance. The values obtained were taken down manually. The results are described in the report.

DETERMINATION OF BODY WEIGHTS
- The body weights was recorded on day 1 (prior to dosing) and prior to sacrifice (pre-test) or prior to treatment with 3HTdR (main experiment).

INTERPRETATION OF RAW DATA
- The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) 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/animal 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:
(i) 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.
(ii) 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 DATA
- 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 2017 (see Annex 1 and 2, attached).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

GENERAL CALCULATIONS
- The mean values and standard deviations were calculated in the body weight tables.
- An EC3 value could not be calculated according to the equation EC3 = (a-c) [(3-d)/(b-d)] + c since all values obtained were above the threshold index of 3 (where 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).
- An approximate EC3 value was derived by means of extrapolation (Gerberick et. al. 2004, Cockshott et. al. 2006).
- All calculations conducted on the DPM values were performed with a validated test script of “R”, a language and environment for statistical computing and graphics.
- Within the program the Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers. An outlier (DPM value determined for animal 2) was detected in the Grubb’s, but not in the Dean-Dixon-Test, and was therefore not excluded from any calculations.

Results and discussion

Positive control results:

The S.I. determined for the positive control was 8.1, demonstrating the validity of the study.

In vivo (LLNA)

Results

Key result

Parameter:

EC3

Value:

1.48

Remarks on result:

other: derived value

Cellular proliferation data / Observations:

RESULTS
- Individual data are given in Table 1 (attached).
- Calculation of stimulation indices per dose group is shown in the table below.

CALCULATION OF THE EC3 VALUE
- The EC3 value could not be calculated.
- Since all S.I. values were above the threshold value of 3, an estimate EC3 value was derived by means of extrapolation.

VIABILITY / MORTALITY
- No deaths occurred during the study period.

CLINICAL SIGNS
- No signs of systemic toxicity were observed during the study period.
- From day 1 to 6, the animals showed an erythema of the ear skin (Score 1 to 3, see details in Appendix 2, attached).

BODY WEIGHTS
- Individual body weight values are shown in Appendix 3 (attached).
- 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.

EAR THICKNESS
- Individual data are included in Appendix 4 (attached).
- The measured ear thickness of all animals treated was recorded daily. A biologically relevant increase in ear thickness was not observed.

Any other information on results incl. tables

CALCULATION OF STIMULATION INDICES PER DOSE GROUP

Test item concentration	Group calculation Mean DPM per animal (2 lymph nodes)*	Group calculation SD	Group calculation S.I.
Vehicle Control Group (cotton seed oil)	5802.0	2111.5	1.0
2.5 % test item	28172.4	4010.9	4.9**
5 % test item	42694.0	7219.8	7.4**
10 % test item	33396.0	3720.0	5.8**
Vehicle Control Group for the Positive Control Item (acetone/olive oil (4+1, v/v))	981.6	574.0	1.0
Positive Control Group (25% α -HCA)	7943.8	2750.0	8.1**

* Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals)

** Statistically significant versus respective vehicle control

Applicant's summary and conclusion

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Conclusions:

The test item was found to be a skin sensitiser. By means of extrapolation, an approximate EC3 value of 1.48 % was derived.

Executive summary:

GUIDELINE

An investigation was performed to assess the skin sensitisation potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The study was conducted in compliance with OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 22 July 2010) and Commission Regulation (EC) No. 440/2008, B.42: “Skin Sensitisation: Local Lymph Node Assay”, updated 06 July 2012.

 

METHODS

The test item, formulated in cotton seed oil, 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 achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment.

 

RESULTS

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. From day 1 to 6, the animals showed an erythema of the ear skin. A biologically relevant increase in ear thickness was not observed. Stimulation Indices (S.I.) of 4.9, 7.4, and 5.8 were determined with the test item at concentrations of 2.5, 5, and 10 % in cotton seed oil, respectively. A clear dose response was not observed. The S.I. for the animals treated with the positive control was 8.1, demonstrating the validity of the assay.

 

CONCLUSION

The test item was found to be a skin sensitiser. By means of extrapolation, an approximate EC3 value of 1.48 % was derived.