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Administrative data

Description of key information

Not sensitising to skin, OECD 429, EU Method B.42 (mouse), Sanders (2012)

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:
14 June 2012 to 28 June 2012
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:
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:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: CBA/Ca (CBA/CaOlaHsd)
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 23 g
- Housing: the animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum access to mains tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25°C
- Humidity (%): 30 - 70%
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): lighting was controlled by a time switch to give twelve hours continuous light (0600 to 1800) and twelve hours darkness.

IN-LIFE DATES: From: 14 June 2012 To: 28 June 2012
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
100 (undiluted test material), 50 and 25 % v/v
No. of animals per dose:
5 animals per dose
Details on study design:
PRELIMINARY SCREENING TEST
A preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the undiluted test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre-dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

MAIN TEST
- Test Material Administration
The mice were treated by daily application of 25 µL of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of five mice received the vehicle alone in the same manner.

- 3H-Methyl Thymidine Administration
Five days following the first topical application of the test material or vehicle (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmol) giving a total of 20 µCi to each mouse.

- Observations
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

- Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group, the draining auricular lymph nodes were excised and processed. For each individual animal, 1 mL of PBS was added to the lymph nodes.

Preparation of Single Cell Suspension: A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish. The lymph node cells suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 x gravity) for ten minutes. The pellet was resuspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 mL of 5% trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation: After approximately eighteen hours incubation at approximately 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 x gravity) for ten minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by β-scintillation counting. The "Poly Q" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

INTERPRETATION OF RESULTS
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).

The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitiser".
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett's Multiple Comparison test was used and for non-homogenous datasets Dunnett's T3 Multiple Comparison Method was used.
Positive control results:
The positive control was administered at 25% v/v in acetone/olive oil 4:1 and produced a stimulation index of 5.76. This corresponds to a positive result.
Therefore, the positive control was found to be a sensitiser under the conditions of the test.
Parameter:
SI
Value:
1.66
Test group / Remarks:
25% v/v
Parameter:
SI
Value:
2.2
Test group / Remarks:
50% v/v
Parameter:
SI
Value:
1.96
Test group / Remarks:
100% v/v
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Disintegrations per minute are displayed in Table 1. The highest dpm was found in the 50 % v/v group, which had a dpm of 7060.18.
Cellular proliferation data / Observations:
- Preliminary Screening Test
No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted. Based on this information the undiluted test material was dosed at 50% and 25% v/v in acetone/olive oil 4:1 test concentrations in the main study.

- Main Test
None of the animals died during the study and no signs of systemic toxicity were noted in either test or control animals. Bodyweight changes in the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.
Disintegrations per minute and stimulation index data provided in Table 1.

Table 1 Individual Disintegrations per Minute and Stimulation Indices

Concentration (% v/v) in Acetone/Olive Oil 4:1

Animal Number

dpm/Animal

Mean dpm/Animal (Standard Deviation)

Stimulation Index

Result

 

 

Vehicle

1-1

2844.07

 

 

3215.81

(± 1053.38)

 

 

-

 

 

-

1-2

2487.28

1-3

4167.09

1-4

2095.85

1-5

4484.76

 

 

25

2-1

5160.23

 

 

5343.08*

(± 342.36)

 

 

1.66

 

 

Negative

2-2

5027.72

2-3

5834.42

2-4

5129.12

2-5

5563.90

 

 

50

3-1

7561.57

 

 

7060.18***

(± 1486.09)

 

 

2.20

 

 

Negative

3-2

9262.66

3-3

6185.95

3-4

6949.54

3-5

5341.17

 

 

100

4-1

6624.77

 

 

6302.47**

(± 1243.43)

 

 

1.96

 

 

Negative

4-2

6495.92

4-3

7974.24

4-4

5862.80

4-5

4554.61

†Total number of lymph nodes per animal is 2

* Significantly different from the control group (p < 0.05)

** Significantly different from the control group (p < 0.01)

*** Significantly different from the control group (p < 0.001)

Interpretation of results:
GHS criteria not met
Conclusions:
The test material was considered to be a non-sensitiser under the conditions of this study in accordance with EU criteria.
Executive summary:

The potential of the test material to act as a sensitiser was investigated in accordance with the standardised guidelines OECD 429 and EU Method B.42 under GLP conditions.

The study assessed the skin sensitisation potential of the test material in female mice of the CBA/Ca strain following topical application to the dorsal surface of the ear. Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of the undiluted test material or the test material as a solution in acetone/olive oil 4:1 at concentrations of 50 or 25%. A further group of five animals was treated with acetone/olive oil 4:1 alone. A concurrent positive control test, using a group of five animals, was performed with α-hexylcinnamaldehyde at a concentration of 25 % v/v in acetone/olive oil 4:1.

The results for all three concentrations of the test material were negative, whilst the positive control material gave the expected result.

Therefore, the test material was considered to be a non-sensitiser under the conditions of this study in accordance with EU criteria.

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

The key study assessed the skin sensitisation potential of the test material in female mice of the CBA/Ca strain following topical application to the dorsal surface of the ear. Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of the undiluted test material or the test material as a solution in acetone/olive oil 4:1 at concentrations of 50 or 25 %. A further group of five animals was treated with acetone/olive oil 4:1 alone. A concurrent positive control test, using a group of five animals, was performed with α-hexylcinnamaldehyde at a concentration of 25 % v/v in acetone/olive oil 4:1.

The results for all three concentrations of the test material were negative, whilst the positive control material gave the expected result.

Therefore, the test material was considered to be a non-sensitiser under the conditions of this study in accordance with EU criteria.

 

The first supporting study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch (1997).

The study was performed to a method comparable to the Guinea Pig Maximisation Test described in the current OECD 406 guideline with some minor deviations. The animals were induced intradermally and topically with a 10 % solution of the test material and later challenged with a 5, 10, 20, or 40 % concentration. A negative control and a positive control were also conducted in a similar manner. Some reactions were noted at the 40 % concentration; however based on the criteria provided in the study to be considered a positive response, under the conditions of the test, the authors concluded that the test substance is negative for skin reactions up to a concentration of 20 %. Although some reactions were noted at the challenge stage in the 40 % concentration group, these did not reach the score defined in the methodology as the level to be considered a positive response both in the positive response and mean response values.

 

The second supporting study was awarded a reliability score of 3 in accordance with the criteria of Klimisch (1997) due to some fundamental flaws in the experimental design.

The sensitisation potential of the test material was assessed in a Maximisation Study in guinea pigs. The method was reportedly "Skin Sensitisation" Magnusson and Kligman Maximization Test. Five animals were used in each of the exposed and control groups. The animals were induced first intradermally and then topically at a concentration of 10 %, then challenged topically with concentrations of 40, 20, 10 and 5 % w/v. The reactions were recorded and classified. Under the conditions of the test, the authors did not observe any sensitisation in Hartley guinea pigs tested with concentrations of 5, 10, 20 and 40 % w/v of the test material in Maximisation Test. The test material was classified as a non-sensitiser in the guinea pig up to a concentration of 40 %.

Migrated from Short description of key information:

One key study and two supporting studies are provided. The key study is an LLNA test and the supporting studies are Maximisation tests. All studies return a negative result, showing the test material to be a non-sensitiser.

Justification for selection of skin sensitisation endpoint:

The key study was conducted in line with GLP and in accordance with the standardised guidelines OECD 429 and EU Method B.42. It was well reported and adhered to sound scientific principles. It was awarded a reliability score of 1 in accordance with the criteria of Klimisch (1997).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation 1272/2008, the test material does not require classification for skin sensitisation.