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:
22 February 2017 to 07 March 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
various deviations to study plan with no impact on results or integrity of the study (see below)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
various deviations to study plan with no impact on results or integrity of the study (see below)
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
Details on test material:
- Appearance/physical state: Light brown, slightly 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: 12 to 13 weeks; Main study: 7 to 8 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: ethanol/water (3+7, v/v)
Concentration:
25, 50 and 100 % test item
No. of animals per dose:
Five
Details on study design:
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 100% of the undiluted test item. Test item solution at different concentrations was prepared using ethanol/water (3+7, v/v) as vehicle. Vortexing 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, 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 50 and 100% 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, attached).
- At the tested concentrations the animals did not show any signs of systemic toxicity. From day 1 to 6, the animals showed an erythema of the ear skin (Score 1 to 2, see Appendix 1, attached). Additionally, bald spots behind the ears were observed (see Appendix 1, attached).
- Thus, the test item in the main study was assayed at 25, 50, and 100 %. 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 AND ANALYSIS
- The test item was placed into a volumetric flask on a tared balance and ethanol/water (3+7, v/v) was quantitatively added (w/v).
- The formulations of the different test item concentrations were prepared serially.
- The preparations were made freshly before each dosing occasion.

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 25, 50, and 100% in ethanol/water (3+7, v/v). 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).

3H-METHYL THYMIDINE ADMINISTRATION
- 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 euthanized 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 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.

OBSERVATIONS
- 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.
- Determination of body weights: The body weights were 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.

GENERAL CALCULATIONS
- The mean values and standard deviations were calculated in the body weight tables.
- Where appropriate, the EC3 value were 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.
- 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 19) was detected in the Grubb’s Test but not in the Dean-DixonTest and was therefore not excluded from calculations.
- Biological and statistical significance were considered together.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
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 October 2016 (see Annex 1 and 2 (attached)).

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
0.7
Remarks on result:
other: 25 % test item in ethanol/water (3+7 v/v)
Key result
Parameter:
SI
Value:
1.3
Remarks on result:
other: 50 % test item in ethanol/water (3+7 v/v)
Key result
Parameter:
SI
Value:
1.3
Remarks on result:
other: 100 % test item in ethanol/water (3+7 v/v)
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 below the threshold value of 3.

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

CLINICAL SIGNS
- Signs of systemic toxicity were not observed during the study period. The animals treated with test item concentrations of 50 and 100 % showed an erythema of the ear skin (Score 1 to 2) on several days (see Appendix 2, attached). A
- Animals treated with 25 % test item concentration did not show any signs of local skin irritation.

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 from day 1, prior to the 1st application, to day 6, prior to necropsy. A 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

(ethanol/water (3+7, v/v))

1382.3

480.4

1.0

25 % test item

922.7

195.1

0.7

50 % test item

1747.9

250.2

1.3

100 % test item

1834.1

366.2

1.3

Vehicle Control Group for the Positive Control Item (acetone/olive oil (4+1, v/v))

2339.3

1050.1

1.0

Positive Control Group (25% α-HCA)

14768.3**

4984.1

6.3

* 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 (p < 0.05)

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be a non-sensitiser under the conditions of the study.
Executive summary:

GUIDELINE

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 ethanol/water (3+7, v/v) was assessed for its possible skin sensitising potential. For this purpose a local lymph node assay was performed using test item concentrations of 25, 50 (w/v) and 100 %. Three groups each of five female mice were treated with different concentrations of the test item by topical application at the dorsum of each ear once daily each on three consecutive days. Three further groups of five mice each were treated with the positive control item or the respective vehicle for the positive control item (acetone:olive oil (4+1 v/v)) or for the test item (ethanol/water (3+7, v/v)). Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine (3H-methyl thymidine; 3HTdR). Approximately five hours after intravenous injection, the mice were sacrificed and the draining auricular lymph nodes excised and pooled per animal. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells were then determined by the incorporation of 3H-methyl thymidine measured in a β-scintillation counter.

 

RESULTS

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. The animals treated with test item concentrations of 50 and 100% showed an erythema of the ear skin (Score 1 to 2) on several days (see Appendix 2 for details). Animals treated with 25 % test item concentration did not show any signs of local skin irritation. A relevant increase in ear thickness was not observed. In this study Stimulation Indices (S.I.) of 0.7, 1.3, and 1.3 were determined with the test item at concentrations of 25, 50 (w/v) and 100 % in ethanol/water (3+7, v/v), respectively. The S.I. of the positive control was 6.3.

 

CONCLUSION

The test item was considered to be a non-sensitiser under the conditions of the study.