Ethyltriphenylphosphonium iodide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-01-21 to 2008-06-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2002

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2004

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Ethyltriphenylfosfonium bromide

EC Number:

216-223-3

EC Name:

Ethyltriphenylfosfonium bromide

Cas Number:

1530-32-1

Molecular formula:

C20H20P.Br

IUPAC Name:

bromo(ethyl)triphenylphosphorane

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Species: Mouse, CBA strain, inbred, SPF-Quality
- Age at study initiation: approx. 10 weeks
- Weight at study initiation: 19 - 24 g
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 - 23.7
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

1, 2.5 and 5%

No. of animals per dose:

5

Details on study design:

- Compound solubility:
-- Vehicle: Propylene glycol (Merck, Darmstadt, Germany)
-- Rationale: The vehicle was selected based on trial formulations performed at the testing laboratory and on test substance data supplied by the sponsor.
-- Preparation: The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.
-- Test substance concentrations selected for the main study were based on the results of a preliminary study. At a 1, 2.5 and 5% test substance concentration no severe irritation was observed.
- Dose selection: Test substance concentrations selected for the main study were based on the results of a preliminary study.
- Ear thickness measurements: no data
- Grading of skin reactions: Erythema and edema were scored similar to the grading system stipulated in OECD Guideline 404

MAIN STUDY

Induction - Days 1, 2 and 3:
The dorsal surface of both ears was epidermally treated (25 µL/ear) with the test substance concentration, at approximately the same time per day. The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing. The control animals were treated the same as the experimental animals, except that, instead of the test substance, the vehicle alone was administered.

Treatment· Day 6:
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 µCi of 3H-methyl thymidine (GE Health care, Buckinghamshire, UK). After approximately five hours, all animals were killed by intraperitoneal injection with pentobarbital Euthesate® (0.2 mL/animal) (Ceva Sante Animale BV, Naaldwijk, The Netherlands). The draining (auricular) Iymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

Tissue processing for radioactivity - Day 6:
A single cell suspension of Iymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 1251-1m). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4° C. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) at 4° C during the night.

Radioactivity measurements - Day 7:
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinEImer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ±0.2% or a maximum of 5 minutes whichever comes first The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Observations:
- Mortality/Viability: Twice daily.
- Toxicity: At least once daily.
- Body weights: On Days 1 (pre-treatment) and 6.
- Necropsy: The animal found dead in the main study was subjected to necropsy for gross macroscopic examination.
- Irritation: On Day 3 (3-4 hours after treatment), the skin reactions were assessed. Erythema and edema were scored simuiar to the grading system stipulated in OECD Guideline 404.

Interpretation:
DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group.
If the results indicate a SI >= 3, the test substance may be regarded as a skin sensitiser, based on the test guideline and recommendations done by ICCVAM , NIH publication; No 99-4494, February 1999
The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations and the EC criteria for classification and labeling of dangerous substances and preparations (Council Directive 67/548/EEC and all adaptations to technical progress and amendments of this Directive published in the Official Journal of the European Communities). Consideration was given to the EC3 value (the estimated test substance concentration that will give a SI =3) (Basketter et al., Appl ToxicoI1999;19:261-266).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:

The six monthly reliability check with Hexylcinnamaldehyde, indicates that the Local Lymph Node Assay as performed at testing laboratory is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

1%

Results 2

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

2.5%

Results 3

Parameter:

SI

Test group / Remarks:

5%

Remarks on result:

not measured/tested

Remarks:

due to mortality/toxicity (refer to 'Any other information on results')

Any other information on results incl. tables

Preliminary irritation study:

At a 10 and 25% test substance concentration, piloerection, ptosis, uncoordinated movements, squeaking when touched, hypothermia and/or flat posture were noted and the animals were sacrificed in extremis at Day 2. At a 1, 2.5 and 5% test substance concentration no signs of systemic toxicity were noted and no severe irritation was observed.

Based on the results, the highest test substance concentration selected for the main study was a 5% concentration.

Main study:

At Day 3, one animal at 5% was found dead before scoring of the skin reactions. Macroscopic post mortem examination of the animal found dead did not reveal any abnormalities. Based on the observed mortality at 5% in the main study and taking the observed toxicity at 10 and 25% in the preliminary irritation study into account, the results of the group at 5% could not be used for interpretation.

- Skin reactions / Irritation:

No skin reactions were observed in any of the control animals and animals at 1 and 2.5%.

- Macroscopy of the auricular Iymph nodes and surrounding area:

One node of one control animal was considered enlarged in size and both nodes of one animal at 2.5% were considered reduced in size. The other nodes of controI animals and animals at 1 and 2.5% were considered normal in size. No macroscopic abnormalities of the surrounding area were noted.

- Body weights:

Body weights and body weight gain of experimental animals at 1 and 2.5% remained in the same range as controls over the study period.

- Radioactivity measurements:

The median DPM/animal values are reported due to possible outlier responses for individual animals within groups.

Median DPM/animal values for the experimental groups treated with test substance concentrations 1 and 2.5% were 412 and 417 respectively. The median DPM/animal value for the vehicle control group was 403.

- Toxicity and Mortality:

No mortality occurred and no symptoms of systemic toxicity were observed in control animals and animals at 1 and 2.5% of the main study.

- Conclusion:

Based on the observed mortality at 5% in the main study and taking the observed toxicity at 10 and 25% in the preliminary irritation study into account, the results of the group at 5% could not be used for interpretation.

The SI values calculated for the substance concentrations 1 and 2.5% were 1.0 and 1.0 respectively.

Since there was no indication that the test substance could elicit an SI >=3 when tested up to 2.5%, it was established that the EC3 value (if any) exceeds 2.5%.

The six monthly reliability check with Hexylcinnamaldehyde, indicates that the Local Lymph Node Assay as performed at the test laboratory is an appropriate model for testing for contact hypersensitivity.

Based on these results:

According to the recommendations made in the test guidelines, the test item is not regarded as skin sensitizer.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The SI values calculated for the substance concentrations 1 and 2.5% were 1.0 and 1.0 respectively. Since there was no indication that the test substance could elicit an SI >=3 when tested up to 2.5%, it was established that the EC3 value (if any) exceeds 2.5%.
Based on these results:
- according to the recommendations made in the test guidelines, Ethyltriphenylfosfonium bromide is not regarded as skin sensitizer.
- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2004), Ethyltriphenylfosfonium bromide does not have to be classified for sensitization by skin contact.
- according to the criteria of REGULATION (EC) No 1272/2008, Ethyltriphenylfosfonium bromide does not have to be classified and has no obligatory labeling requirement for sensitization by skin contact.