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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:
09 December 2012 to 05 February 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
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
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: CBA/J Rj mice
- Age at study initiation: Main Test: 9 weeks old (age-matched, within one week); Preliminary Test 8 week old mice.
- Weight at study initiation: Main Test: 20.1 - 21.6 g (the weight variation in animals in the study did not exceed ± 20 % of the mean weight); Preliminary Test: 20.3 - 21.1 g.
- Housing: In groups in type II polypropylene / polycarbonate cages.
- Diet: Complete diet for rats/mice was provided ad libitum.
- Water: Tap water from the municipal supply was provided ad libitum.
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 15-20 air exchange/hour.
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Vehicle:
other: 1 % Pluronic in distilled water
Concentration:
25, 10 and 5 % (w/v)
No. of animals per dose:
Four per dose, including both negative and positive controls.
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The maximum concentration of test material in an acceptable solvent was established according to OECD guideline 429. The following standard OECD vehicles were assessed: AOO (acetone:olive oil 4:1 (v:v) mixture), N,N-dimethylformamide, Methyl ethyl ketone, Propylene glycol, Dimethyl sulfoxide and 1 % aqueous Pluronic® PE9200 (1% Pluronic). The best vehicle taking into account the test material characteristics and its usage was considered to be 1 % Pluronic. The highest achievable concentration based on the regulatory requirements of the OECD guideline and the physical characteristics of the test material was, 25 (w/v) %.
- Irritation: No irritation was observed in the preliminary test, detailed in the field “any other information on materials and methods incl. tables”.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response: The test material is regarded as a sensitizer if both of the following criteria are fulfilled:
> That exposure to at least one concentration of the test material resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
>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.
- Negative control: Four animals were treated with the vehicle only (1 % Pluronic).

TREATMENT PREPARATION AND ADMINISTRATION
Preparation: The test material was weighed and formulations prepared daily on a weight:volume basis (as (w/v) %).
Administration: animals were topically dosed with 25 μL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Parameter:
SI
Remarks on result:
other: The stimulation index values were 1.6, 0.8 and 0.9 at concentrations of 25, 10 and 5 (w/v) %, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The DPM values were were 2201.0, 1105.0 and 1255.0 at concentrations of 25, 10 and 5 (w/v) %, respectively. The DPM per node (DPN) values were were 275.1, 138.1 and 156.9 at concentrations of 25, 10 and 5 (w/v) %, respectively.

OBSERVATIONS

- Clinical observations: No mortality or signs of systemic toxicity were observed during the study. There were no indications of any irritancy at the site of application. Test mateiral precipitate was observed in the 25 (w/v) % dose group on Days 1-5 and in the 10 and 5 (w/v) % dose groups on Days 2-3.

- Bodyweight measurement: No treatment related effects were observed on body weight.

- Lymph nodes: The appearance of the lymph nodes was normal in the negative (vehicle) control group and in all test item treated groups. Larger than normal lymph nodes were observed in the positive control group.

 

INTERPRETATION OF OBSERVATIONS

Since, there were no confounding effects of irritation or systemic toxicity at the applied concentrations, the proliferation values obtained are considered to reflect the real potential of the test material to cause lymphoproliferation in the Local Lymph Node Assay. The resulted stimulation indexes observed under these exaggerated test conditions were considered to be good evidence that the test material is a non-sensitizer (see Figure 1).

POSITIVE CONTROL RESUTLS

No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A significant lymphoproliferative response (stimulation index value of 5.4) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.

Furthermore, the DPN values observed for the vehicle and positive control substance in this experiment were within the historical control range. Each treated and control group included 4 animals.

 

Table 2: DPM, DPN and Stimulation Index Values for all Groups

Test Group

Measured DPM/group

DPM

No. of Lymph Nodes

DPN

Stimulation Index

Background

(5 (w/v) % TCA)

30

-

-

-

-

Negative Control

1442

1412.0

8

176.5

1.0

Test Material

25 (w/v) %

2231

2201.0

8

275.1

1.6

Test Material

10 (w/v) %

1135

1105.0

8

138.1

0.8

Test Material

5 (w/v) %

1285

1255.0

8

156.9

0.9

Positive Control

7721

7691.0

8

961.4

5.4

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the test, no mortality, systemic clinical signs, treatment related effects on body weights, or indications of any irritancy at the site of application were observed in any of the treatment groups. Test material precipitate was observed in the 25 (w/v) % dose group on Days 1-5 and in the 10 and 5 (w/v) % dose groups on Days 2-3. The observed stimulation index values were 1.6, 0.8 and 0.9 at concentrations of 25, 10 and 5 (w/v) %, respectively. Since the measured indices were less than 3 the test material is considered to be a non-sensitizer.
Executive summary:

The skin sensitisation potential of the test material was determined in a GLP study conducted in accordance with the standardised guidelines OECD 429 and EU Method B. 42, using the local lymph node assay method.

In the definitive, test animals were administered the test material at 25, 10 and 5 (w/v) %, formulated in 1 % Pluronic aqueous solution. The dosing range and vehicle were selected based on the results of two preliminary studies, a compound solubility test and a preliminary irritation/toxicity test. Negative vehicle and positive controls (97.8 % hexyl cinnamic aldehyde) were run concurrently for comparison.

Four female CBA/J Rj mice per dose were topically administered 25 µL of the appropriate test material formulation on the dorsal surface of each ear for three consecutive days (days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (³HTdR) and the values obtained were used to calculate stimulation indices (SI).

Under the conditions of the test, no mortality, systemic clinical signs, treatment related effects on body weights, or indications of any irritancy at the site of application were observed in any of the treatment groups. Test material precipitate was observed in the 25 (w/v) % dose group on Days 1-5 and in the 10 and 5 (w/v) % dose groups on Days 2-3. The observed stimulation index values were 1.6, 0.8 and 0.9 at concentrations of 25, 10 and 5 (w/v) %, respectively. Since the measured indices were less than 3, the test material is considered to be a non-sensitizer.

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

In the key study (Váliczkó, 2013), the skin sensitisation potential of the test material was determined in a GLP study conducted in accordance with the standardised guidelines OECD 429 and EU Method B. 42, using the local lymph node assay method.

In the definitive, test animals were administered the test material at 25, 10 and 5 (w/v) %, formulated in 1 % Pluronic aqueous solution. The dosing range and vehicle were selected based on the results of two preliminary studies, a compound solubility test and a preliminary irritation/toxicity test. Negative vehicle and positive controls (97.8 % hexyl cinnamic aldehyde) were run concurrently for comparison.

Four female CBA/J Rj mice per dose were topically administered 25 µL of the appropriate test material formulation on the dorsal surface of each ear for three consecutive days (days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).

Under the conditions of the test, no mortality, systemic clinical signs, treatment related effects on body weights, or indications of any irritancy at the site of application were observed in any of the treatment groups. Test material precipitate was observed in the 25 (w/v) % dose group on Days 1-5 and in the 10 and 5 (w/v) % dose groups on Days 2 - 3. The observed stimulation index values were 1.6, 0.8 and 0.9 at concentrations of 25, 10 and 5 (w/v) %, respectively. Since the measured indices were less than 3, the test material is considered to be a non-sensitizer.


Migrated from Short description of key information:
Non-sensitizer, OECD 429, EU Method B. 42, LLNA, Váliczkó 2013.

Justification for selection of skin sensitisation endpoint:
The available study was performed in accordance with standardised guidelines and under GLP conditions. The study was performed to a good standard with a sufficient level of reporting to assess the data provided. In accordance with the principles of assessing data quality the study was assigned a reliability score of 1 (Klimisch 1997). The study was therefore considered sufficient to address this endpoint.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Skin Sensitisation

According to the criteria outlined in Regulation (EC) No. 1272/2008, the test material does not meet the criteria for classification as a skin sensitizer.