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Description of key information

Skin sensitisation (OECD 429): not 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:
26 Aug - 23 Sep 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
no ear thickness measurement in the pre-screen test
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
23 Jul 2010
Deviations:
yes
Remarks:
No ear thickness measurement in the pre-screen test
GLP compliance:
yes (incl. QA statement)
Remarks:
The Department of Health of the Government of the United Kingdom
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, Bicester, UK
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8 - 12 weeks
- 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 (Harlan Teklad, Bicester, UK), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): Approximately 15
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
other: Butanone
Concentration:
25, 50 and 100% (v/v)
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS:
1 female mouse was treated by daily application of 25 μl of the undiluted test substance to the dorsal surface of the ear, for 3 consecutive days. The body weight was recorded on Day 1 prior to dosing and on Day 6.
- Systemic toxicity: The animal was observed for signs of toxicity twice on Day 1 and 2, and once on Day 4, 5 and 6. The body weight was recorded on Day 1 prior to dosing and on Day 6.
Irritation: The animal was observed for local skin irritation to the application site twice on Day 1 and 2, and once on Day 4, 5 and 6.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by β-scintillation and γ-counting
- Criteria used to consider a positive response: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) 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”.
- Other: The animals were observed for signs of toxicity twice on Day 1 and 2, and once on Day 4, 5 and 6. The body weight was recorded on Day 1 prior to dosing and on Day 6 prior to termination.

TREATMENT PREPARATION AND ADMINISTRATION: 25 µl of the test material was applied to the entire dorsal surface of each ear of each mouse on Day 1, 2 and 3 in concentrations 25% and 50% in butanone, and undiluted. The irritation effects on the treatment site were assessed daily. On Day 6 an injection of 250 µl phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine (3H-TdR) was made into the tail vein of each experimental mouse. Five hours later, the draining auricular lymph node of each ear was excised into PBS and pooled per experimental group. A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze and rinsed with PBS. The precipitates were incubated for approximately 18 h at approximately 4 °C, centrifuged, resuspended in 1 mL TCA and transferred to 10 mL scintillation fluid before β-scintillation counting.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
A study (study dates: 16 - 22 Apr 2009; project number: 0039/1083) was performed to assess the sensitivity of the strain of mouse used at the testing facility to a known sensitizer and show the sensitivity and reproducibility of the test. The positive control substance hexyl cinnamic aldehyde (15% (v/v) in butanone) was considered to be a sensitizer under the conditions of the test (SI 4.08).
Key result
Parameter:
SI
Value:
1.16
Test group / Remarks:
25% (v/v)
Key result
Parameter:
SI
Value:
1.56
Test group / Remarks:
50% (v/v)
Key result
Parameter:
SI
Value:
1.47
Test group / Remarks:
100% (v/v)
Cellular proliferation data / Observations:
CLINICAL OBSERVATIONS: No mortality and no signs of systemic toxicity were noted in the test or control animals during the test. Fur loss was noted in 4/4 animals treated with undiluted test material or the test material at a concentration of 50% (v/v). The area of the fur loss was not reported.

DETAILS ON STIMULATION INDEX CALCULATION
The SI of the 25, 50 and 100% treatment group was 1.16, 1.56 and 1.47, respectively. None of the test substance concentrations produced a 3-fold increase in 3HTdR incorporation.

EC3 CALCULATION
None of the SI values were above 3 and it is therefore not possible to determine a EC3 concentration.

BODY WEIGHTS: Body weight changes were comparable to those observed in the corresponding control group animals over the same period.

Table 1: Individual body weights and the results of the radioactive disintegrations

Concentration in butanone (% (v/v))

Body weight (g)

Radioactivity incorporated

Day 1

Day 6

Gains

DPM

dpm/node

SI

Control

18

18

0

9834.09

1229.26

-

19

19

0

21

20

-1

21

22

1

25

18

18

0

11428.97

1428.62

1.16

20

21

1

17

16

-1

20

20

0

50

20

20

0

15303.39

1912.92

1.56

19

19

0

22

22

0

19

19

0

100

19

19

0

14486.57

1810.82

1.47

21

20

-1

21

20

-1

20

21

1

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The skin sensitising potential of the test substance was evaluated in a Local Lymph Node Assay according to OECD 429 and in compliance with GLP (Key, 2009). The study was conducted on four female mice per dose group (25%, 50% and 100%). Each animal received 25 µL of the test substance (diluted in butanone where applicable) to the dorsum of each ear. Animals were treated once daily for three days. After a two-day rest period, all animals were injected with tritiated methyl-thymidine in the tail vein. Five hours later, animals were sacrificed, and the draining auricular lymph nodes removed and pooled per experimental group. A single cell suspension of pooled lymph node cells was prepared and the lymph node cell proliferation measured via beta-scintillation counting. A vehicle control (butanone) group and a positive control group (15% alpha-hexylcinnamaldehyde in butanone) of four females each were run concurrently, and verified that the test system is reliable. No mortality and no signs of systemic toxicity was observed in any animal. Fur loss was noted in 4/4 animals treated with undiluted test substance and 50% dilution, but no skin irritation was observed. The simulation index (SI) of the 25, 50 and 100% treatment group was 1.16, 1.56 and 1.47, respectively. The test substance produced a stimulation index smaller than 3 in all groups and is therefore not considered as skin sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available data on skin sensitisation of the test substance do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.