Registration Dossier

Administrative data

Description of key information

- LLNA, Skin sensitizer (OECD 429, GLP, K, Rel. 2)
- Not sensitising in humans at 1% (HRIPT).

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:
January 20 to February 11, 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP Study performed according to the old version of the OECD test guideline No. 429 (2002), therefore ear thickness measurements were not included in the pre-screen test. The substance being moderately irritating to the skin, this deviation may have an impact on the reliability of the study results. However no visible signs of irritation were observed at any of the concentration tested, therefore the study results are considered as reliable.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Adopted on 24 April 2002
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 Monitoring Programme (inspected on August 19, 2008/ signed on March 4, 2009)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: Animals were individually housed, in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: Food (2014C Teklad Global Rodent diet supplied by Harlan Teklad., Oxon, UK), ad libitum
- Water: Mains tap water, ad libitum.
- Acclimation period: 5 days

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

IN-LIFE DATES: From: January 20, 2009 To: February 11, 2009
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Main test: 10, 25 and 50 % v/v in acetone/olive oil 4:1
No. of animals per dose:
4 females
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Undiluted test item was used (100 %). Soluble at 50% in AOO 4:1.
- In a preliminary screening test, one mouse/dose were treated by daily application of 25 µL of the undiluted test material or the test material at a concentration of 50 % v/v in acetone/olive oil 4:1, to the dorsal surface of each ear for up to three consecutive days (Days 1, 2, 3). The mice were observed twice daily on Day 1, pre-dose on Day 2 and the surviving mouse post-dose on Day 2, twice on Day 3 and once daily on Days 4, 5 and 6. Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and of the surviving mouse on Day 6. The bodyweight of the mouse that was humanely killed was recorded immediately prior to termination.
- Irritation: The animal treated with the undiluted test material was humanely killed, pre-dose on Day 2, due to the occurrence of clinical signs of toxicity (hunched posture, lethargy and splayed gait). No signs of skin irritation were noted. No signs of systemic toxicity or skin irritation were noted in the animal treated with the test material at a concentration of 50 % v/v in acetone/olive oil 4:1. Based on this information the dose levels selected for the main test were 10, 25 and 50 % v/v in acetone/olive oil 4:1.

MAIN STUDY
- Name of test method: Local Lymph Node Assay
TREATMENT PREPARATION AND ADMINISTRATION:
Groups of four mice were treated with the test material at concentrations of 10, 25 and 50 % v/v in acetone/olive oil 4:1. 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). A further group of four mice received the vehicle alone in the same manner. Each animal was injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 20 μCi of 3H-methyl thymidine on Day 6. After five hours, all animals were killed by carbon dioxide asphyxiation and the draining (auricular) lymph node of each ear was excised. The nodes from the four mice were excised and pooled for each experimental group in 1 mL PBS. A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle mechanical disaggregation through gauze. LNC were washed with PBS by centrifugation at 1400 rpm (approximately 190 g) for 10 minutes and pooled lymph node cells were pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 mL of 5 % Trichloroacetic acid (TCA). After approximately 18 h incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) 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 number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

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).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No data
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 15 % v/v. A further control group of five animals was treated with acetone/olive oil 4:1 alone. With a SI = 6.55, α-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitiser under the conditions of the test.
Key result
Parameter:
EC3
Value:
> 2
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Mean DPM / animal for vehicle, 10, 25 and 50 % v/v in acetone/olive oil 4:1 were 8971.92, 14744.66, 38972.35 and 88885.89, respectively.

DETAILS ON STIMULATION INDEX CALCULATION
- Stimulation index for 10, 25 and 50 % v/v in acetone/olive oil 4:1 were 1.64, 4.34 and 9.91, respectively.
- A stimulation index of greater than 3 was recorded for the test material at concentrations of 25 and 50 % v/v in acetone/olive oil 4:1.
- A stimulation index of less than 3 was recorded for the test material at a concentration of 10 % v/v in acetone/olive oil 4:1.

EC3 CALCULATION
EC3 > 2%

CLINICAL OBSERVATIONS
There were no deaths. No signs of systemic toxicity or skin irritation 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 7.4.1/1: Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

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

dpm

dpm/Node

Stimulation Index

Result

Vehicle

8971.92

1121.49

na

na

10

14744.66

1843.08

1.64

Negative

25

38972.35

4871.54

4.34

Positive

50

88885.89

11110.74

9.91

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

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the test conditions, test material is classified as skin sensitisers (Category 1B) according to the annex VI of 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 with the undiluted test material in which hunched posture, lethargy and splayed gait were observed and lead to humane killing of the animal, a second test was conducted at 50 % v/v. No clinical signs of toxicity were not at 50% v/v, this concentration was therefore selected as the highest dose investigated in the main test.

Three groups, each of four females, were treated with 50 μL (25 μL per ear) of test material as a solution in acetone/olive oil 4:1 at concentrations of 10, 25 and 50 % v/v for 3 consecutive days. A further group of four animals was treated with acetone/olive oil 4:1 alone. The animals were allowed to rest without dosing on days 4 and 5. On day 6, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of 3H-Methyl Thymidine.

 

Mean DPM / animal for vehicle, 10, 25 and 50 % v/v in acetone/olive oil 4:1 were 8971.92, 14744.66, 38972.35 and 88885.89, respectively. Stimulation index for 10, 25 and 50 % v/v in acetone/olive oil 4:1 were 1.64, 4.34 and 9.91, respectively. A stimulation index of greater than 3 was recorded for the test material at concentrations of 25 and 50 % v/v in acetone/olive oil 4:1. A stimulation index of less than 3 was recorded for the test material at a concentration of 10 % v/v in acetone/olive oil 4:1.There were no deaths. No signs of systemic toxicity or skin irritation were noted in the test or control animals during the test. 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 historical positive control, α-Hexylcinnamaldehyde, gave a SI of 6.55, when tested at 15 % v/v. The test system was therefore considered to be valid.

 

Under the test conditions, test material is classified as skin sensitizer (Category 1B) according to the annex VI of the Regulation EC No. 1272/2008 (CLP) and to the GHS since EC3 is higer than 2%.

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, 2009, rel.2). This Local Lymph Node Assay was conducted according to the OECD test guideline No 429 and in compliance with GLP.

Following a preliminary screening test with the undiluted test material in which hunched posture, lethargy and splayed gait were observed and lead to humane killing of the animal, a second test was conducted at 50 % v/v. No clinical signs of toxicity were not at 50%, this concentration was therefore selected as the highest dose investigated in the main test.

Three groups, each of four females, were treated with the test material as a solution in acetone/olive oil 4:1 at concentrations of 10, 25 and 50 % v/v for 3 consecutive days. A further group of four animals was treated with acetone/olive oil 4:1 alone.

The historical positive control, α-Hexylcinnamaldehyde, gave a SI of 6.55, when tested at 15 % v/v. The test system was therefore considered to be valid.

Stimulation index for 10, 25 and 50 % v/v in acetone/olive oil 4:1 were 1.64, 4.34 and 9.91, respectively.

There were no deaths. No signs of systemic toxicity or skin irritation were noted in the test or control animals during the test. 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.

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

A human repeated insult patch test in human has also been performed with the substance at up to 1 % (TKL, 2010). The study was concluded to be negative for sensitisation.

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 > 2%.

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