Registration Dossier

Administrative data

Description of key information

Skin sensitizer 1B (OECD 429, GLP, K, rel.1)

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:
23 October 2012 to 09 January 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 429 without any deviation.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 22 July 2010
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:
UK GLP Compliance Programme (inspected on July 10, 2012/ signed on November 30, 2012)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Limited, Oxon, UK.
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: Aimals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet: Food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK), ad libitum
- Water: Mains tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: Approximately 15 changes/h
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: 23 October 2012 to 09 January 2013
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Initial test: 25 and 50% v/v in acetone/olive oil 4:1 and 100% (undiluted test item)
Additional test: 5% v/v in acetone/olive oil 4:1
No. of animals per dose:
5 females/dose
Details on study design:
PREPARATION OF TEST ITEM:
For the purpose of the study, the test item was used undiluted and freshly prepared as a solution in acetone/olive oil 4:1. This vehicle was chosen as it produced the most suitable formulation at the required concentration. The test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.

PRE-SCREEN TESTS:
- Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μl of the undiluted test item 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 to 3 and Days 1 to 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.
- Results: 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 item and the test item at concentrations of 50% and 25% v/v in acetone/olive oil 4:1 were selected for the main test.

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 mice were treated by daily application of 25 μl of the appropriate concentration of the test item and vehicle control to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3) using a micropipette. The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre-dose and post dose on Day 1 and on Days 2 to 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between Days 1 to 3 and Days 1 to 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.
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 μl of sterile phosphate buffered saline (PBS) containing tritiated 3H-methyl thymidine (3HTdR:80μCi/ml, specific activity 2.0 Ci/mmol, ARC UK Ltd) giving a total of 20 μCi to each mouse.
Five hours later animals were killed by carbon dioxide asphyxiation followed by cervical separation. The lymph nodes from each group were pooled and a single cell suspension was prepared. Cells were washed with phosphate buffered saline (PBS) and precipitated with 5% trichloroacetic acid (TCA) for 18 h at ca. 4 °C. The pellets were recovered by centrifugation and resuspended in 1 mL of TCA and transferred to a vial containing scintillation fluid. 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 20 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).
Additional Test: In order to determine the concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value), and at the request of the Study Sponsor, an additional group of five animals was treated with the test item at a concentration of 5% v/v in acetone/olive oil 4:1.An additional group of five mice received the vehicle alone in the same manner. These animals were treated and observed as described in the initial test.

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:
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.
Probability values (p) are presented as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
P≥0.05 (not significant)
Positive control results:
A group of five animals was treated with 50 µl (25 µl per ear) of α-Hexylcinnamaldehyde, tech., 85% as a solution in acetone/olive oil 4:1 at a concentration of 25 % v/v. A further control group of five animals was treated with acetone/olive oil 4:1 alone. With a SI = 6.29, α-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitiser under the conditions of the test.
Key result
Parameter:
SI
Remarks:
additional test
Value:
2.12
Test group / Remarks:
5% v/v in acetone/olive oil 4:1
Remarks on result:
other: negative
Key result
Parameter:
SI
Value:
6.19
Test group / Remarks:
25% v/v in acetone/olive oil 4:1
Remarks on result:
other: positive
Key result
Parameter:
SI
Value:
13.93
Test group / Remarks:
50% v/v in acetone/olive oil 4:1
Remarks on result:
other: positive
Key result
Parameter:
SI
Value:
15.06
Test group / Remarks:
100%
Remarks on result:
other: positive
Key result
Parameter:
EC3
Value:
9
Test group / Remarks:
25% v/v in acetone/olive oil 4:1
Remarks on result:
other: positive
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX: The Stimulation Index values expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows: 2.12, 6.19, 13.93 and 15.06 at 5, 25 and 50% v/v in acetone/olive oil 4:1. See table 7.4.1/1

EC3 CALCULATION:
EC3 = c + [[(3−d)/(b−d)] x (a−c)]
a: 25; b: 6.19; c: 5; d: 2.12
EC3 = 10 + [[(3−1.24)/(3.81−1.24)] x (25−10)] = 9
The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 9%.

CLINICAL OBSERVATIONS: There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. No visual local skin irritation or excessive irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted at any dose concentration evaluated.

BODY WEIGHTS: Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Table 7.4.1/1: Individual disintegrations per minute and stimulation indices – main test – initial and additional test

Concentration (% v/v) in

acetone/olive oil 4:1

Animal

Number

dpm/

Animal a

Mean dpm/Animal

(Standard Deviation)

Stimulation

Index b

Result

Initial test

Vehicle

1-1

2001.55

2371.39 (±595.04)

NA

NA

1-2

3098.80

1-3

1585.83

1-4

2729.80

1-5

2440.99

25

2-1

13906.46

14685.11** (±3745.96)

6.19

Positive

2-2

12722.18

2-3

21170.76

2-4

13952.57

2-5

11673.60

50

3-1

31532.74

33023.91*** (±4411.90)

13.93

Positive

3-2

28850.98

3-3

38819.66

3-4

36466.86

3-5

29449.30

100

4-1

33151.62

35721.32*** (±4145.47)

15.06

Positive

4-2

33690.94

4-3

37902.03

4-4

41947.57

4-5

31914.46

Additional test

Vehicle

1-1

723.13

2279.81 (±2360.18)

na

na

1-2

1359.86

1-3

1571.17

1-4

6464.46

1-5

1280.43

5

2-1

3049.49

4838.93 (±1554.49)

2.12

Negative

2-2

4605.40

2-3

5325.23

2-4

4027.40

2-5

7187.12

dpm = Disintegrations per minute

a = Total number of lymph nodes per animal is 2

b = Stimulation Index of 3.0 or greater indicates a positive result

na = Not applicable

** = Significantly different from control group p<0.01

*** = Significantly different from control group p<0.001

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the test conditions, test item is classified as a contact sensitiser (Category 1B) according to the Regulation EC No. 1272/2008 (CLP) and to the GHS.
Executive summary:

A study was performed to assess the skin sensitisation potential of test material in the CBA/Ca (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 which no clinical signs of toxicity were noted at a test item concentration of 100%, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 μl (25 μl per ear) of the undiluted test item or the test item as a solution in acetone/olive oil 4:1 at concentrations of 50% or 25% v/v. A further group of five animals was treated with acetone/olive oil 4:1 alone. A further group of five animals was treated with acetone/olive oil 4:1 alone. In order to determine the concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value), and at the request of the Study Sponsor, an additional group of five animals was treated with the test item at a concentration of 5% v/v in acetone/olive oil 4:1. An additional group of five animals was treated with acetone/olive oil 4:1 alone.

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. No visual local skin irritation or excessive irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted at any dose concentration evaluated. Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

The Stimulation Index values expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows: 2.12, 6.19, 13.93 and 15.06 at 5, 25 and 50% v/v in acetone/olive oil 4:1 and 100%, respectively. The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 9%.

The historical positive control, α-Hexylcinnamaldehyde, gave a SI of 6.29, when tested at 25 % v/v. The test system was therefore considered to be valid.

Under the test conditions, test item is classified as a contact sensitiser (Category 1B) according to the Regulation EC No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for sensitisation endpoint.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

A key study was identified (Harlan, 2013). In this dermal sensitisation study performed according to the OECD guideline No. 429 and in compliance with GLP, the test material was tested in the CBA/Ca strain mouse. Following a preliminary screening test in which no clinical signs of toxicity were noted at the substance concentration of 100%, three groups, each comprising five mice receiving test item at concentrations of 25, 50% or 100% v/v. Similarly constituted groups received the vehicle 4:1 v/v acetone: olive oil (AOO) or positive control substance (25% v/v hexyl cinnamic aldehyde). At the request of the study sponsor, an additional group of five mice was treated with the test item at a concentration of 5% v/v. Mice were treated by daily application of 25µL of the appropriate concentration or control (vehicle or positive), to the dorsal surface of both ears for three consecutive days.The proliferative response of the lymph node cells (LNC) from the draining auricular lymph nodes was assessed five days following the initial application, by measurement of the incorporation of 3H-methyl Thymidine (3HTdR) by β-scintillation counting of LNC suspensions. The response was expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio), termed as Stimulation Index (SI).

The SI obtained for 5, 25, 50 and 100% v/v were 2.12, 6.19, 13.93 and 15.06, respectively, which indicates that test item showed the potential to induce skin sensitization. The EC3 value was calculated to be 9% v/v. No sign of systemic toxicity or excessive local skin irritation were noted at the concentrations of 5, 25, 50 and 100% v/v.

The SI for the positive control substance hexyl cinnamic aldehyde was 6.29, which demonstrates the validity of this study.

 

Under the test conditions, test material is classified as a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Under the REACH regulation there is no legal standard information requirement in Annexes VII to X to perform any specific test for respiratory sensitisation. In addition, no validated or widely recognised in vitro or in vivo test methods specific to respiratory sensitisation are available yet. No human or animal data are available on the substance to address respiratory sensitisation. No alert for respiratory sensitisation were found by the OECD QSAR Toolbox.

Justification for classification or non-classification

Harmonized classification:

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

Self-classification:

Based on the available data, the substance is classified as Skin Sens. 1B, H317 (May cause an allergic skin reaction) according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS, since EC3 is > 2% (9%).

No direct scientific data are available on the substance to address respiratory sensitisation.