Registration Dossier

Administrative data

Description of key information

OECD 429; GLP; concentrations: 3%, 10%, 30%; not sensitizing (1999).

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
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)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan UK, Blackthorne, Bicester, Oxon, UK
- Age at study initiation: young adults
- Housing: cages suitable for animals of this strain and weight range (max. of 4 mice per cage)
- Diet (e.g. ad libitum): R&M No.1 (Special Diet Services Limited, Witham, Essex, UK), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: The animals were housed under the experimental conditions for at least 5 days, prior to the start of the study .

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): A minimum of 15
- Photoperiod (hrs dark / hrs light): 12 / 12
Vehicle:
propylene glycol
Concentration:
the test substance was applied as 3%, 10% or 30% w/v preparations in propylene glycol
No. of animals per dose:
4
Details on study design:
TREATMENT PREPARATION AND ADMINISTRATION:
Groups of four male mice were used for this study. Approximately 25μ1 of a 3%, 10% or 30% w/v preparation of the test substance in propylene glycol was applied, using a variable volume micro-pipette, to the dorsal surface of each ear. A vehicle control group was similarly treated using propylene glycol alone. The procedure was repeated daily for 3 consecutive days. A concurrent naïve control group was not treated with the test substance or the vehicle .
Three days after the third application, all the animals were injected, via the tail vein, with approximately 250 μl of phosphate buffered saline (PBS) containing approximately 20 μCi of a 2.OCi/mmol specific activity 3H-methyl thymidine . Approximately 5 hours later, the animals were humanely killed by inhalation of halothane vapour followed by cervical dislocation. The draining auricular lymph nodes were removed from each animal and, together with the nodes from the other animals in the group, were placed in a container of PBS .
A single cell suspension was prepared by mechanical disaggregation of lymph nodes through a 200-mesh stainless steel gauze . The cell suspensions were then washed three times by centrifugation with approximately 10 ml of PBS. Approximately 3 ml of 5% w/v trichloroacetic acid (TCA) was added and after overnight precipitation at 4°C, the samples were pelleted by centrifugation and the supernatant was discarded. The cells were then resuspended in approximately 1 ml of TCA .
The lymph node suspensions were transferred to scintillation vials and 10 mL of scintillant (Optiphase) was added prior to ß-scintillation counting using a Packard Tri-Carb 2500TR Liquid Scintillation Counter.

CLINICAL OBSERVATIONS:
Animals were checked at least once daily for signs of systemic toxicity.

CRITERIA USED TO CONSIDER A POSITIVE RESPONSE:
The criterion for a positive response is that one or more concentrations of the test substance should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The application of hexylcinnamaldehyde at concentrations of 1%, 3% and 10% w/v in acetone resulted in a greater than 3-fold increase in isotope incorporation at the 10% w/v concentration. Therefore, hexylcinnamaldehyde was shown to be a skin sensitiser, confirming the validity of the protocol used for the study.
Parameter:
SI
Value:
>= 1.16 - <= 1.61
Remarks on result:
other: 3% w/v: 1.16 10% w/v: 1.61 30% w/v: 1.31
Parameter:
other: disintegrations per minute (DPM)
Value:
>= 2 322 - <= 3 216
Remarks on result:
other: naïve control: 2301 0 (vehicle only): 1989 3% w/v: 2322 10% w/v: 3216 30% w/v: 2613

The application of the test substance at concentrations of 3%, 10% and 30% w/v in propylene glycol resulted in an increase in isotope incorporation which was less than 3-fold at all three concentrations. Consequently, the test substance is designated as unlikely to be a moderate or strong sensitiser under the conditions of the test.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions chosen, the test substance did not show any skin sensitizing potential and thus, was concluded not to be a skin sensitizer.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In a GLP-compliant LLNA study conducted according to OECD guideline 429, groups of four male CBA/Ca mice per dose level were treated with a 3%, 10% or 30% w/v preparation in propylene glycol or with the vehicle alone (CTL, 1999). Each test animal was applied with 25 µL of the test substance preparation to the dorsum of both ears for three consecutive days. Three days after the last application the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring the proliferative capacity of the prepared lymph node cells. The application of the test substance at concentrations of 3%, 10% and 30% w/v in propylene glycol resulted in an increase in isotope incorporation which was less than 3-fold at all three concentrations (SI indices were: 1.16 (3%); 1.61 (10%); 1.31 (30%)). Therefore, the test substance is considered to be not sensitizing.

In three further LLNA assays according to OECD guideline 429 and in compliance with GLP, three additional members of the same category were analyzed for their sensitization potential (see attached category justification). None of the tests gave a positive response, therefore all tested substances were considered as not sensitizing.

Conclusion: Based on the available data for the test substance and taking the data of category members into account, no classification for sensitization is warranted. The test substance is considered to be not a sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the data, classification for sensitization is not warranted under Regulation (EC) No.1272/2008.