Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-03-27 - 2015-05-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP and OECD 442B-compliant study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Deviations:
yes
Remarks:
(see below)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
yes
Remarks:
(see below)
Qualifier:
according to
Guideline:
other: NIH No. 99-4494 (The Murine Local Lymph Node Assay)
Version / remarks:
A Test Method for Assessing The Allergic Contact Dermatitis Potential of Chemical/Compounds
Deviations:
yes
Remarks:
(see below)
Principles of method if other than guideline:
Due to the severe toxicity observed after the first dose, the screen was terminated and all animals sacrificed on Day 2.
Body weights were mesured on Day 6 prior to euthanasia, not prior to BrdU injection on Day 5. The animals were weighed at the proper time (Day 6) as per guidelines.
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Remarks:
Homogenous Liquid
Specific details on test material used for the study:
Trade name: CYPHOS IL 101 Phosphonium Salt
Physical state: liquid
Composition of test material, percentage of components: Trihexyl(tetradecyl)phosphonium chloride (258864-54-9), > 95%
Lot/batch No.: WEC031280
Expiration date of the lot/batch: >1 year (March 2016) when stored at room temperature and protected from direct contact with water (hydrophobic)

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Jackson Laboratories (Bar Harbor, ME)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: The mice were 8 weeks of age at the start of the primary irritancy study and 9 weeks of age at the start of the LLNA.
- Weight at study initiation: The Day 1 body weight range for the Screen animals was 17.8 - 20.0 grams; and for the Quantitative Irritation Test (QIT) animals was 17.3 - 21.0 grams; and for the Main Test animals was 17.3 - 22.1 grams. The weight variation of the animals at study start did not exceed +/- 20% of the mean body weight.
- Housing: The animals (5/group) were housed one per cage in suspended wire-bottom cages. Bedding was placed beneath the cages and changed at least three times per week.
- Diet: Fresh PMI Rodent Chow (Diet #5001), ad libitum
- Water: available ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): temperature-controlled
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0.05%, 0.1% and 0.25%
No. of animals per dose:
5 (female)
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility:
The test article was tested for solubility in Acetone:Olive Oil (4:1) and found to be soluble at 25% and 50% (v/v), and AOO was chosen as the vehicle for the study.

- Preliminary Dermal Irritation Screen:
Treatment of increasing concentration (10%, 25% and 50% (v/v)) in AOO was made by topical application to the dorsum of each ear on Day 1. The test article was spread over the entire dorsal surface of the ear using a micropipette at 25µL/ear. Due to severe toxicity observed after the first dose and at the request of the Sponsor, the screen was terminated and all animals were sacrificed on Day 2.

- Quantitative Irritation Test (QIT):
Six groups of two CBA/J mice per group were treated with six concentrations (0.25%, 0.5%, 1%, 2.5%, 5.0% and 10% v/v) of the test article in AOO. Treatment was made by topical application to the dorsum of each ear once daily for three consecutive days. The test article was spread over the entire dorsal surface of the ear using a micropipette at 25µL/ear.

- Lymph node isolation and processing:
In the QIT, gross observations of the auricular lymph nodes were made at sacrifice on Day 6. The lymph nodes were not collected.

MAIN STUDY
The test article concentrations used in the main LLNA study were chosen such that the maximum concentration tested was the highest achievable solution of the test article in the vehicle, while avoiding both overt systemic toxicity and excessive local dermal-irritation. Five groups of CBA/J mice (5 animals per group) were treated by topical application of the test article (0.05%, 0.1% and 0.25% (v/v)), vehicle control or positive control in the same manner as in the QIT. AOO was chosen as the vehicle, based on solubility.

Lymph Node Isolation and Processing:
Four days after the first topical application (Day 5) 0.5 ml of BrdU solution (5mg/per mouse/injection) were intraperitoneally injected.
Approximately 24 hours after treatment with BrdU the mice were sacrificed and the draining lymph nodes were rapidly excised and pooled for each animal (2 nodes per animal).

Determination of BrdU Incorporation:
The incorporation of BrdU into lymph node cells was determined using a commercial cell proliferation assay kit as follows (Roche Applied Science, Indianapolis): 100 μl of the lymph node cell suspension were added to the wells of a flat-bottom microplate in triplicate. After fixation and denaturation of the lymph node cells, anti-BrdU antibody was added to each well and allowed to bind. Subsequently the anti-BrdU antibody was removed by washing and the substrate solution was then added and allowed to produce chromogen. After stopping the substrate reaction the absorbance was measured at 370 nm with a reference wavelength of 492 nm (MicroQuant plate reader, Bio-Tek Instruments). The mean optical density (OD) values of three "blank" wells (containing PBS) were used as a reference.

ANIMAL ASSIGNMENT AND TREATMENT
-- Animal assignment:
Animals were stratified using pre-exposure body weights and randomly assigned to treatment groups using a computer program
- Name of test method:
Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response:
Chemicals that elicit a SI >=1.6 in the LLNA are characterized as potential sensitizing substances.

TREATMENT PREPARATION AND ADMINISTRATION:
Test formulations were prepared fresh for each day of dosing. The administration of the materials (25 µl/ear) was made on the dorsal surface of both ears using a micropipette. All mice received one of three concentrations (0.05%, 0.1% and 0.25% in AOO) or AOO alone on days 1-3.

TYPE AND FREQUENCY OF OBSERVATIONS
All animals in the study were observed once daily throughout the study for clinical signs, either of local irritation at the application site or systemic toxicity, and for mortality.
Body weights were recorded on Day 1, immediately prior to dosing, and on Day 6 (prior to sacrifice).
Since mild irritation was noted during the irritation screen, the thickness of each ear was monitored during the LLNA procedure: on Day 1 prior to dosing, on Day 3 before the third test article application and on Day 6 before sacrifice. Changes in ear thickness on Day 3 and Day 6 relative to Day 1 were expressed as a percent of the Day 1 pre-dose values. Ear thickness increases of 25% or more were considered biologically significant (based on the scientific literature and historical laboratory data) and deemed indicative of a greater than moderate local dermal irritation response.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Analysis of Stimulation Index Data:
Calculation of Stimulation Index (SI): For each animal, lymph node cell proliferation was determined by BrdU-ELISA and the mean Optical Density (OD) +/- SD was then calulated for each group. The number of proliferating lymphocytes in each animal was then divided by the mean number of proliferating lymphocytes in the Vehicle Control group, according to the equation below:
Stimulation Index (SI) per animal = Mean OD(test substance)/Mean OD(vehicle)
The mean SI and standard deviation (SD) were calculated for each group.

EC1.6 calculation:
The concentration at which SI = 1.6 (EC1.6) was calculated according to the following equation:
EC1.6 = c+[(1.6-d)/(b-d)]*(a-c)
Where:
a = test article concentration at SI value "b"
b = SI value nearest to but greater than 1.6
c= test article concentration at SI value "d"
d = SI value nearest to but less than 1.6

Statistical analysis:
For each test group, the individual animal SI values along with the mean group SI and standard deviation were calculated, and ANOVA followed by Student's t-Test was run to statically compare each test article dose group to the Vehicle Control group.

Results and discussion

Positive control results:
The 25% HCA SI value was 2.7, SI >= 1.6 indicates a sensitizing response. HCA is a useful positive control for the LLNA as it is a moderate contact sensitizer and can serve as a better indication of the assay's sensitivity compared with extreme sensitizers such as DNCB.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1
Variability:
0.3
Test group / Remarks:
0.05%
Parameter:
SI
Value:
1.1
Variability:
0.6
Test group / Remarks:
0.1%
Parameter:
SI
Value:
2.7
Variability:
0.5
Test group / Remarks:
0.25%
Parameter:
other: EC1.6
Value:
0.14
Remarks on result:
other: expressed in % (v/v)
Cellular proliferation data / Observations:
EAR THICKNESS
In the Main Test, treatment with 0.25% (v/v) of test substance resulted in excessive dermal irritation. (See Table 1)

DETAILS ON STIMULATION INDEX CALCULATION
The mean SI and standard deviation for each treatment group was calculated (Table 2). A result is regarded as a positive response in the LLNA if at least one concentration results in a 1.6-fold or greater increase in LNC proliferation relative to that of Vehicle Control Animals.

EC1.6 CALCULATION
The EC1.6 was calculated to be 0.14% (v/v).

CLINICAL OBSERVATIONS:
In the main test, all animals survived the in-life phase and were observed to be normal.

BODY WEIGHTS
In the main test, body weight losses were noted but were not significant (< 2 grams).

Any other information on results incl. tables

Table 1: Mean Ear Thickeness (mm)

Mean Ear Thickeness (mm) Change (%)
Treatment Pre-Dosing (Day1) 48 Hr (Day 3) End In-Lifr (Day 6) Day 3 - Day 1 Day 6 - Day 1 
AOO (Vehicle Control) 0.19 0.19 0.19 0.0% 0.0%
25% HCA (Postive Control) 0.20 0.23 0.22 15.0% 10.0%
0.05% (v/v) Test Item 0.20 0.20 0.20 0.0% 0.0%
0.1% (v/v) Test Item 0.19 0.20 0.20 5.3% 5.3%
0.25% (v/v) Test Item 0.19 0.22 0.23 22.2% 27.8%a

a = an increase in ear thickeness of 25% indicates a positive dermal irritation response

Table 2: Mean Stimulation Index

Treatment SI S.D.
AOO (Vehicle Control) 1.0 0.6
25% HCA (Postive Control) 2.7a 1.9
0.05% (v/v) Test Item 1.0 0.3
0.1% (v/v) Test Item 1.1 0.6
0.25% (v/v) Test Item 2.7a 0.5

a = SI ≥ 1.6 indicates a sensitizing response

Table 3: Test Article Statistics

Comparison of SI values by Student t-Test Mean Difference P Value
AOO (Vehicle Control) vs. 0.05% (v/v) Test Item -0.02000 >0.05 ns
AOO (Vehicle Control) vs. 0.1% (v/v) Test Item -0.1400 >0.05 ns
AOO (Vehicle Control) vs. 0.25% (v/v) Test Item -1.720 <0.001 ***

*** = statistically significant (p<0.001)

ns = not statistically significant (p>0.05)

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Topical application of Trihexyltetradecylphosphonium chloride at 0.25% (v/v) in AOO resulted in an SI value greater than 1.6 (SI>1.6). Therefore, this test article is a dermal sensitizer in the Local Lymph Node Assay with an EC1.6 of 0.14% (v/v).
Executive summary:

In this study the test item Trihexyltetradecylphosphonium chloride was assessed for its skin sensitizing potential using the Local Lymph Node Assay (LLNA) in mice according to OECD TG 442B and under GLP conditions.

Test item solution at different concentrations was prepared in the vehicle acetone : olive oil (AOO, 4:1, v/v). There were no difficulties in administration of the test article or with its adherence to the dosed ears. On Day 2 of the screen, all animals showed signs of toxicity – edema of the face (cheeks) and sagging eyelids. Both animals in each of the 25% and 50% test article dose groups also appeared emaciated and had few feces. Both animals in 50% test article dose group were also lethargic. All animals lost more than 2 grams of body weight by Day 2. The screen was then terminated and the animals were sacrificed.

In the QIT, both animals in each of the 0.5%, 1%, 2.5% and 5% dose group had erythema on and around the ears beginning on Day 3; this was also observed in both animals in the 10% dose group only on Day 2. Edema on the face and ears was observed in one animal in the 2.5% dose group on Day 5 and 6, both animals in the 5% dose group on Days 4 to 6, and one animal on the 10% dose group on Day 4 to 6. One animal in the 10% dose group had edema of the face (cheeks) on Day 2 and 3, lethargy on Day 2 to 6, appeared emaciated on Day 3 to 6, and few feces and piloerection on Days 4 to 6. The second animal in the 10% dose group had edema of the face (cheeks) and lethargy on Days 2 and 3, few feces and appeared emaciated on Day 3, and was found dead on Day 4; this animal lost more than 2 grams. In the surviving animals, a body weight loss was noted but was not significant (<2 grams).

In the main test, all animals survived the in-life phase and were observed to be normal. Ear thickness measurements and individual animal observations indicated that treatment with 0.25% (v/v) of the test article resulted in excessive local dermal irritation. Body weight losses were noted but were not significant (<2 grams).

The SI of the positive Control Group, 25% HCA, was 2.7. The group SI values for the test article were as follows:

Test article concentration 0.05% (v/v)  0.1% (v/v)  0.25% (v/v) 
SI Value  1.0 1.1 2.7

Topical application of Trihexyltetradecylphosphonium chloride at 0.25% (v/v) in AOO resulted in an SI value greater than 1.6 (SI>1.6). Therefore, the test article is a dermal sensitizer in the Local Lymph Node Assay with an EC1.6 of 0.14% (v/v).