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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 01 to 28, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in compliance with OECD Guideline No. 429 without any deviation.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
inspected on July 13-16, 2015 / signed on September 14, 2015
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc. Postbus 6174, 5960 AD Horst / The Netherlands.
- Age at study initiation: Pre-test and main study: 9-10 weeks
- Weight at study initiation: 20.2 ± 1.3 g
- Housing: Animals were group housed in Makrolon Type II (pre-test) / III (main study), with wire mesh top
- Diet: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 45-65%
- Air changes: at least 8 to 10 air changes per hour
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From July 01 to 28, 2015
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Main test: 25, 50 and 100 % v/v in acetone/olive oil 4:1
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- 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.
- At the tested concentrations the animals did not show any signs of systemic toxicity (including a >5% reduction in bodyweight from Day 1 to Day 6). From day 4 to 6, the animal treated with 100% test item concentration showed a very slight erythema of the ear skin (Score 1) and scaly ear skin on day 6. The animal treated with 50% test item concentration did not show any signs of local skin irritation. Maximum increases in ear thickness in the preliminary screening were 2.1 and 9.9% in the two animals treated with 50 and 100%, respectively.
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.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: The test item will be regarded as a sensitiser if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a ‘non-sensitiser’.

TREATMENT PREPARATION AND ADMINISTRATION: The highest test item concentration, which can 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. The different test item concentrations were prepared individually. The preparations were made freshly before each dosing occasion.
- Each test group of five mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 25, 50, and 100% 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 with a micropipette, using the tip of the pipette. Treatment was conducted once daily for three consecutive days (Days 1, 2, and 3). A further group of five mice (control animals) was treated in the same manner with an equivalent volume of the relevant vehicle alone (control animals). Five days after the first topical application (Day 6) 250 μL of phosphate-buffered saline containing 19.7 μCi of 3H-methyl thymidine (equivalent to 78.9 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein. 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 individual animal (2 nodes per animal). For each individual 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, preferably in darkness. 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.

- The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
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.
The Dean-Dixon-Test and the Grubb’s test were used for identification of possible outliers (performed with validated program R Script Outlier.Rnw). No outlier value was detected.
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. SI for the positive control substance 25% hexyl cinnamic aldehyde (HCA), was 9.48 which demonstrates the validity of this study.
Parameter:
SI
Remarks on result:
other: Stimulation index for vehicle, 25, 50 and 100 % v/v were 1.00, 0.73, 1.81 and 0.99, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM per animal for vehicle, 25, 50 and 100 % v/v were 2023.2 ± 475.5, 1473.8 ± 523.7, 3664.6 ± 1382.7 and 2012.8 ± 1056.2, respectively.

Table 7.4.1/1: Calculation of Stimulation Indices per Dose Group

Test item concentration

 

Group Calculation

 

Result

Mean DPM per animal (2 lymph nodes) a)

 

SD

S.I

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

 

2023.2

475.5

1.00

-

25%

1473.8

523.7

0.73

Negative

50%

3664.6

1382.7

1.81

Negative

100%

2012.8

1056.2

0.99

Negative

 

a) 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)

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

Viability / Mortality: No deaths occurred during the study period.

Clinical Signs: No symptoms of local skin irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period.

Body Weights: 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.

Body weight change of the test animals between Day 1 and Day 6 were comparable to that observed in the corresponding control group animals over the same period.
Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the test conditions, test material is not classified as a skin sensitiser according to the annex VI of the Regulation EC No. 1272/2008 (CLP).
Executive summary:

A study was performed to assess the skin sensitisation potential of test material in the CBA/CaOlaHsd strain mouse following topical application to the dorsal surface of the ear. The method was conducted according to the OECD test guideline No 429 and in compliance with GLP.

 

Following a preliminary screening test in two animals in which no clinical signs of systemic toxicity or excessive local skin irritation were noted at a concentration of 50 and 100%, the latest concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Maximum increases in ear thickness in the preliminary screening were 2.1 and 9.9% in the two animals treated with 50 and 100%, respectively. Therefore this local lymph node assay was performed using test item concentrations of 25, 50, and 100% in groups of five animals each. A further group of animals was treated with the vehicle acetone/olive oil (4+1, v/v) alone.

 

The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed.

 

Mean DPM per animal for vehicle, 25, 50 and 100 % v/v were 2023.2 ± 475.5, 1473.8 ± 523.7, 3664.6 ± 1382.7 and 2012.8 ± 1056.2, respectively. Stimulation Indices (S.I.) of 0.73, 1.81, and 0.99 were determined with the test item at concentrations of 25, 50, and 100% in acetone/olive oil (4+1, v/v), respectively.

 

SI for the positive control substance 25% hexyl cinnamic aldehyde (HCA), was 9.48 which demonstrates the validity of this study.

 

Under the test conditions, test material is not classified as a skin sensitiser according to the annex VI of the Regulation EC No. 1272/2008 (CLP) and of the Directive 67/548/EEC. This study is considered as acceptable and satisfies the requirement for sensitisation endpoint.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

A key study was identified (Envigo, 2015). This Local Lymph Node Assay was conducted according to the OECD test guideline No 429 and in compliance with GLP. Following a preliminary screening test in two animals in which no clinical signs of systemic toxicity or excessive local skin irritation were noted at a concentration of 50 and 100%, the latest concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Maximum increases in ear thickness in the preliminary screening were 2.1 and 9.9% in the two animals treated with 50 and 100%, respectively. Therefore this local lymph node assay was performed using test item concentrations of 25, 50, and 100% in groups of five animals each. A further group of animals was treated with the vehicle acetone/olive oil (4+1, v/v) alone.

Stimulation index for 25, 50 and 100 % v/v in acetone/olive oil 4:1 were 0.73, 1.81, and 0.99 respectively.

The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed.

 

SI for the positive control substance 25% hexyl cinnamic aldehyde (HCA), was 9.48 which demonstrates the validity of this study.

 

Under the test conditions, test material is not classified as a skin sensitiser.

Migrated from Short description of key information:
-LLNA, not a skin sensitizer (OECD 429, GLP, K, Rel. 1)

Justification for selection of skin sensitisation endpoint:
Only one study available. The key-study is GLP-compliant and of high quality (Klimisch score = 1).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008 including ATP7.

Self-classification:

Based on the available data, no additional self-classification is proposed according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP)

No data was available regarding respiratory sensitisation.