Registration Dossier

Administrative data

Description of key information

Skin sensitisation (OECD 429): skin sensitising

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:
16 Feb - 10 Mar 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
It should be noted that there are certain limitations of the LLNA for surfactant type chemicals. Based on the physicochemical properties, PE1 is not considered as a surface-active substance but it is a borderline case and could cause a false-positive result in the LLNA. No analytical purity is given.
Reference:
Composition 0
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)
Remarks:
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
Species:
mouse
Strain:
other: CBA/Ca (CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Limited, Bicester, Oxon, UK.
- Age at study initiation: 8-12 weeks old
- Weight at study initiation: 15-23 g
- Housing: individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet: 2014 Teklad Global Rodent diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxon, UK; ad libitum
- Water: tap water; ad libitium
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
100% (undiluted), 50% and 25% v/v
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
One mouse was treated by daily application of 25 µL of the undiluted test substance to the dorsal surface of each ear for three consecutive days. No signs of systemic toxicity were noted (see table 1). Based on this information, the undiluted test substance and the test substance 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: ³H-methyl thymidine incorporation determined by beta-scintillation
- Criteria used to consider a positive response: The test substance will be regarded as a sensitiser if at least one concentration of the test substance results in a threefold or greater increase in ³HTdR incorporation compared to control values.

TREATMENT PREPARATION AND ADMINISTRATION:
25 μL of the test substance was applied to the entire dorsal surface of each ear of each mouse for three consecutive days (Day 1-3). Local irritation reactions were assessed. On Day 6 an injection of 250 μL phosphate buffered saline (PBS) containing 20 µCi ³H-methyl thymidine (³HTdR; 80 µCi/mL, specific activitiy 2.0. Ci/mmol) was made into the tail vein of each experimental mouse. 5 h later, all mice were sacrificed and the draining auricular lymph nodes were excised into PBS and pooled for each experimental group. A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless stell gauze. The lymph node cells were rinsed through a gauze with 4 mL of PBS into a petri dish. The lymph node cell suspension was transferred to 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 pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 min. The pellet was resuspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was precipitated with 5% trichloroacetic acid at 4 °C for 18 h. ³HTdR incorporation was measured by beta-scintillation counting. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The positive control substance showed a stimulation index of 6.55 (reliability check: historic positive control data, October 2008, project number:0039/1057).
Key result
Parameter:
SI
Value:
22.52
Test group / Remarks:
25% (v/v)
Key result
Parameter:
SI
Value:
25.71
Test group / Remarks:
50% (v/v)
Key result
Parameter:
SI
Value:
23.11
Test group / Remarks:
100%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
The radioactive disintegrations per minute (dpm) are given in Table 2

DETAILS ON STIMULATION INDEX CALCULATION
A stimulation index of greater than 3 was recorded for the undiluted test material and the test material at concentrations of 50% and 25% v/v in acetone/olive oil 4: 1.

EC3 CALCULATION
No EC3 value was established, since no conventional dose-response relationship was observed.

CLINICAL OBSERVATIONS:
There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

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 1: Clinical observations, Body weight and mortality data – preliminary screening test

Concentration

Animal Number

Bodyweight (g)

Day

1

2

3

4

5

6

Day 1

Day 6

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Pre-Dose

Post Dose

100%

S-1

17

19

0

0

0

0

0

0

0

0

0

0= No signs of systemic toxicity

Table 2: Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index (SI)

Concentration
(%v/v) in
acetone/olive oil 4:1

dpm

dpm/Nodea

SIb

Result

Vehicle

6889.00

861.13

na

na

25

155117.80

19389.73

22.52

Positive

50

177120.30

22140.04

25.71

Positive

100

159191.00

19898.88

23.11

Positive

dpm= Disintegrations per minute

a= Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

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

na= Not applicable

Table 3: Individual clinical observations and mortality data

Concentration
(% v/v) in
acetone/olive oil 4:1

Animal Number

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Vehicle

1-1

0

0

0

0

0

0

0

0

0

1-2

0

0

0

0

0

0

0

0

0

1-3

0

0

0

0

0

0

0

0

0

1-4

0

0

0

0

0

0

0

0

0

25

2-1

0

0

0

0

0

0

0

0

0

2-2

0

0

0

0

0

0

0

0

0

2-3

0

0

0

0

0

0

0

0

0

2-4

0

0

0

0

0

0

0

0

0

50

3-1

0

0

0

0

0

0

0

0

0

3-2

0

0

0

0

0

0

0

0

0

3-3

0

0

0

0

0

0

0

0

0

3-4

0

0

0

0

0

0

0

0

0

100

4-1

0

0

0

0

0

0

0

0

0

4-2

0

0

0

0

0

0

0

0

0

4-3

0

0

0

0

0

0

0

0

0

4-4

0

0

0

0

0

0

0

0

0

0= No signs of systemic toxicity

Table 4: Individual body weights and body weight changes

Concentration
(% v/v) in
acetone/olive oil 4:1

Animal Number

Body weight (g)

Body weight Change (g)

Day 1

Day 6

Vehicle

1-1

18

17

-1

1-2

18

18

0

1-3

18

17

-1

1-4

18

18

0

25

2-1

20

19

-1

2-2

18

18

0

2-3

19

19

0

2-4

18

19

1

50

3-1

17

17

0

3-2

17

18

1

3-3

17

17

0

3-4

17

17

0

100

4-1

17

16

-1

4-2

20

19

-1

4-3

20

19

-1

4-4

17

17

0

 


Interpretation of results:
other: Skin Sens Cat 1B is required according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the local lymph node assay, the test substance revealed a SI ≥ 3 at concentrations of 25, 50 and 100%. Therefore, the test substance is considered as weak sensitiser.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The test substance was tested for skin sensitisation properties on mice using the local lymph node assay (Pooles, 2009). The study was conducted according to OECD 429 with the following test concentrations: 25%, 50% or 100% v/v in acetone/olive oil (4:1). A preliminary screening test suggested that the undiluted test substance would not produce systemic toxicity or excessive local irritation. The mice were treated by topical application of the appropriate test concentrations to the dorsal surface of each ear for 3 consecutive days. 5 days following the first application of the test substance, all mice were injected with ³H-methyl thymidine (³HTdR) and sacrificed 5 h afterwards. The draining auricular lymph nodes were excised and pooled for each experimental group. ³HTdR incorporation in lymphocytes was measured by beta-scintillation counting. There were no deaths during the study period and no signs of toxicity or local skin irritation were noted.

The mean stimulation index values were 22.52, 25.71 and 23.11 at treatment concentrations of 25, 50 and 100%, respectively.

Under the test conditions, the test substance was considered to be a skin sensitiser. No EC3 value was established, since no conventional dose-response relationship was observed. Since all SI values were greater than 3, the substance was considered to be a skin sensitiser.

With respect to the positive result of the local lymph node assay, it should be noted that there are certain limitations of the LLNA for surfactant type chemicals. Based on the physicochemical properties (surface tension), PEMB is not considered as a surface-active substance but it is a borderline case and could cause a false-positive result in the LLNA.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available data on skin sensitisation meets the criteria for classification according to Regulation (EC) 1272/2008, and is therefore classified as skin sensitiser Category 1B (H317).