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Administrative data

Description of key information

Results of a local lymph node assay showed the test item to be non-sensitising (OECD 429 and EU Method B.42).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 Febraury 2017 to 07 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
various 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 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)
Species:
mouse
Strain:
other: 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: 4 females.
- Number of animals for the main study: 25 females.
- Number of animals per group: 5 females (nulliparous and non-pregnant).
- Number of test groups: 3
- Number of control (vehicle) groups: 1
- Number of positive control groups: 1
- Age (beginning of treatment): 1st pre-test: 14 to 15 weeks; 2nd pre-test: 15 to 16 weeks; main study: 10 to 13 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: see deviation); 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.
Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
Acetone 99+ %; Olive oil highly refined, low acidity
Concentration:
5, 10, and 25%
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 acetone/olive oil (4+1, 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 5, the animals showed an erythema of the ear skin (Score 1 to 2, see Appendix 1 (attached) for details). Additionally, visible swelling of the ears (only the animal treated with the undiluted test item), slight erythema of the scalp, and an increase of ear weight above the threshold of 25% were observed (see Appendix 1 (attached) for details).
- Therefore, a second pre-test was performed with concentrations of 10 and 25%. From day 1 to 5, the animals showed an erythema of the ear skin (Score 1 to 2, see Appendix 1 (attached) for details). Additionally, eyelid closure, swollen cheeks, and slight erythema of the scalp were observed (see Appendix 1 (attached)for details). However, no indication of excessive local skin irritation as defined by OECD 429 was observed.
- Thus, the test item in the main study was assayed at 5, 10, and 25%. 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 acetone/olive oil (4+1, v/v) was quantitatively added (w/v).
The different test item concentrations were prepared individually.
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 5, 10, and 25% in acetone/olive oil (4+1, 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 (approximately 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)
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).
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
5 % test item
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
10 % test item
Key result
Parameter:
SI
Value:
1.8
Test group / Remarks:
25 % test item
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 showed an erythema of the ear skin (Score 1) on several days (see Appendix 2 (attached) for details).
- Additionally, the animals treated with 25 % test item and the positive control showed slight visible swelling of the ears on days 4 and 5.

BODY WEIGHTS
- Individual body weight values are included 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.

CALCULATION OF STIMULATION INDICES PER DOSE GROUP

Concentration

Group calculation

Mean DPM per animal (2 lymph nodes)*

Group calculation

SD

Group calculation

S.I.

Vehicle Control Group (acetone/olive oil (4+1, v/v))

2339.3

1050.1

1.0

5 % test item

2386.1

1622.4

1.0

10 % test item

2341.9

1346.1

1.0

25 % test item

4149.7

1883.9

1.8

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

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

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 Method B.42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008.

 

METHODS

In order to study a possible skin sensitising potential of the test item, three groups each of five female mice were treated once daily with the test item at concentrations of 5, 10, and 25% in acetone/olive oil (4+1, v/v) by topical application to the dorsum of each ear for three consecutive days. Two further groups each of five mice were treated with the vehicle (acetone:olive oil (4+1 v/v)) only or with the positive control at 25%. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, 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 was 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 showed an erythema of the ear skin (Score 1) on several days. Additionally, the animals treated with 25% test item and the positive control showed slight visible swelling of the ears on days 4 and 5. A significant increase in ear thickness was not observed in any group. Stimulation Indices (S.I.) of 1.0, 1.0, and 1.8 were determined with the test item at concentrations of 5, 10, and 25% in acetone/olive oil (4+1, 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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

A key 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 Method B.42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008.

 

Three groups each of five female mice were treated once daily with the test item at concentrations of 5, 10, and 25% in acetone/olive oil (4+1, v/v) by topical application to the dorsum of each ear for three consecutive days. Two further groups each of five mice were treated with the vehicle (acetone:olive oil (4+1 v/v)) only or with the positive control at 25%. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, 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 was determined by the incorporation of 3H-methyl thymidine measured in a β-scintillation counter.

 

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 showed an erythema of the ear skin (Score 1) on several days. Additionally, the animals treated with 25% test item and the positive control showed slight visible swelling of the ears on days 4 and 5. A significant increase in ear thickness was not observed in any group. Stimulation Indices (S.I.) of 1.0, 1.0, and 1.8 were determined with the test item at concentrations of 5, 10, and 25% in acetone/olive oil (4+1, v/v),respectively. The S.I. of the positive control was 6.3. The test item was considered to be a non-sensitiser under the conditions of the study.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

LLNA results determined the test item to be non-sensitising (Stimulation Index < 3) and, in accordance with ECHA Guidance on the Application of the CLP Criteria (Version 5.0; July 2017), classification as a skin sensitiser is not required under the terms of Regulation (EC) No 1272/2008.