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Toxicological information

Skin sensitisation

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Administrative data

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:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): p-Methoxybenzaldehyde
- Physical state: Colourless/ yellowish liquid
- Analytical purity: 99.71%
- Lot/batch No.: 55
- Sample: Y14020/001
- Expiration date of the lot/batch: 14th March 2007
- Storage condition of test material: Ambient temperature in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd., Manston Road, Margate, Kent, UK
- Age at study initiation: 8-12 weeks
- Housing: A maximum of 4 mice was housed per cage, in cages suitable for animals of this strain and weight range. Environmental enrichment provided included tents, bases and nestlets.
- Diet: RM1, supplied by Special Diet Services Limited, Witham, Essex, UK; ad libitum
- Water: mains water, supplied by an automatic system; ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+-3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
other: 1:3 ethanol:diethylphthalate (1:3 EtOH:DEP)
Concentration:
0 (vehicle control), 1, 2.5, 5, 10, 25%
No. of animals per dose:
4
Details on study design:
Animals were injected, via the tail vein, with approximately 250 µL of phosphate buffered saline (PBS) containing 20µCi of a 2.0Ci/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 10mL of PBS. Approximately 3mL 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 10mL of scintillant (Optiphase) was added prior to beta-scintillation counting using a Packard Tri-Carb 3100TR Liquid Scintillation Counter.


Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The estimated concentration of the test material required to produce a 3-fold increase in the draining lymph node cell proliferative activity (EC3) was calculated by interpolating between two SI points using the following equation; EC3 = [(3 – d)/(b – d)] x (a – c) + c, where a = concentration with SI immediately above 3, b = SI of a, c = concentration with SI immediately below 3, and d = SI of c.

Results and discussion

Positive control results:
Application of hexylcinnamaldehyde at concentrations of 5%, 10% or 25% resulted in an SI of 1.7, 2.3 or 6.4, respectively, confirming the validity of the study protocol.

In vivo (LLNA)

Results
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The application of the test substance at concentrations of 1, 2.5, 5, 10 or 25 %w/v in 1:3 EtOH:DEP resulted in an isotope incorporation which was less than 3-fold at all concentrations: 1%: the test material was not considered a skin sensitizer at 1% with a stimulation index of 1.4. 2.5%: the test material was not considered a skin sensitizer at 2.5% with a stimulation index of 1.0. 5%: the test material was not considered a skin sensitizer at 5% with a stimulation index of 1.1. 10%: the test material was not considered a skin sensitizer at 10% with a stimulation index of 1.5. 25%: the test material was not considered a skin sensitizer at 25% with a stimulation index of 1.5.

Any other information on results incl. tables

Concentration (% w/v) Nr of lymph nodes assessed DPM DPM per lymph node SI
0,0 8 2816 352 -
1,0 8 4066 508 1.4
2,5 8 2795 349 1.0
5,0 8 3152 394 1.1
10,0 8 4136 517 1.5
25,0 8 4322 540 1.5

The EC3 value is estimated to be greater than 25% (6250 µg/cm2).

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Irritancy scoring

The application of the test substance at concentrations of 1, 2.5, 5, 10 or 25 %w/v in 1:3 EtOH:DEP resulted in some signs of irritancy (mild irritation) to the skin on or around the ear area on days 3 and 6 for all four animals of the 10 and 25 %w/v dose groups, respectively.

Applicant's summary and conclusion