Phosphoric acid, C4 and C12-14(even-numbered) Alkyl Esters, Ammonium Salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 June - 19 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Dipotassium butyl phosphate

EC Number:

247-585-0

EC Name:

Dipotassium butyl phosphate

Cas Number:

26290-70-0

Molecular formula:

C4H11O4P.2K

IUPAC Name:

dipotassium butyl phosphate

Constituent 2

Reference substance name:

Potassium dibutyl phosphate

EC Number:

246-749-9

EC Name:

Potassium dibutyl phosphate

Cas Number:

25238-98-6

Molecular formula:

C8H19O4P.K

IUPAC Name:

potassium dibutyl phosphate

Constituent 3

Reference substance name:

Dibutyldipotassium pyrophosphate

Molecular formula:

C8H20O7P2.2K

IUPAC Name:

Dibutyldipotassium pyrophosphate

Test material form:

solid

Specific details on test material used for the study:

Batch/Lot #: 7675229
Manufacture date: August 2015
Expiry date: August 2018
Storage conditions: Controlled Room Temperature (15-25°C, below 70 % RH)

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo, San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF at arrival
- Age at study initiation: 11 weeks old
- Weight at study initiation: 21.3 - 22.7 g
- Housing: Group caging / mice were provided with glass tunnel-tubes. Cage Type II. polypropylene / polycarbonate.
- Diet:ad libitum; ssniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice”
- Water: ad libitum; tap water from the municipal supply
- Acclimation period: 21 days
- Indication of any skin lesions: none

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0 - 25.6°C
- Humidity (%): 30 - 87 %
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

other: 1% aqueous Pluronic® PE9200

Concentration:

Preliminary Irritation/Toxicity Test: 50, 25 %w/v
Main test: 25, 10 or 5 %w/v

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The solubility of the test item was examined in a short Preliminary Compatibility Test. The following standard OECD vehicles were assessed: AOO (acetone:olive oil 4:1 (v:v) mixture), N,N-dimethylformamide (DMF), Methyl ethyl ketone (MEK), Propylene glycol (PG), Dimethyl sulfoxide (DMSO) and 1% aqueous Pluronic® PE9200. The formulations using the listed vehicles at 100 % (w/v) concentrations were not achievable. The best vehicle taking
into account the test item characteristics, its usage and the requirements of the relevant OECD guideline was considered to be 1% Pluronic. The formulations at 50 % (w/v) using 1% Pluronic as vehicle was the highest concentration which was suitable for the test.
- Irritation: The Preliminary Irritation/Toxicity Test was started according to the Study Plan on CBA/CaOlaHsd mice using two doses (2 animals/dose) at test item concentrations of 50% (w/v) and 25 % (w/v) in 1% Pluronic. The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and the radioactive proliferation assay was not performed. Both ears of each mouse were observed for erythema and scored
- Systemic toxicity: In the Preliminary Irritation / Toxicity Test, all mice were observed daily for any clinical signs of systemic toxicity
- Ear thickness measurements: Ear thickness was measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals.
- Erythema scores: Very slight erythema (score 1) was observed for both animals of the 50% (w/v) dose group on Days 1-3.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA (Proliferation assay)
- Criteria used to consider a positive response:
The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
- That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
- The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
FORMULATION:
Following the prelimary compatibility test, the test item was weighed and formulations prepared daily on a weight:volume basis (as % (w/v)) in the Pharmacy of CiToxLAB Hungary Ltd.
TOPICAL APPLICATION:
During the study, animals were topically dosed with 25 μL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.
PROLIFERATION ASSAY:
- Injection of Tritiated Thymidine (3HTdR)
On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS (phosphate buffered saline) containing approximately 20 μCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).
- Removal and Preparation of Draining Auricular Lymph Nodes
Five hours (± 30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses).
The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels.
Once removed, the nodes of mice from each test group was pooled and collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.
- Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). Pooled LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 °C. After centrifugation supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for each group of pooled lymph nodes.
- Determination of Incorporated 3HTdR
After the final washing step, supernatants were removed. Pellets were gently agitated resuspended and 3 mL of 5 % (w/v) TCA solution was added to the tubes for precipitation of macromolecules.
After overnight (approximately 18 hours) incubation at 2-8 °C, precipitates were centrifuged (approximately 190 x g for 10 minutes at 4oC), and supernatants were removed. Pellets were resuspended in 1 mL of 5 % (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).
The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5 % (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.
OBSERVATIONS
- Clinical Observations
During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained.
- Measurement of Body Weight
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.
- Ear thickness measurements
The ear thickness values of the experimental animals were determined by using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6 in the study.
Additional quantification of the ear thickness was also performed in the study by ear punch weight determination on Day 6 after the animals were euthanized.
EVALUATION OF RESULTS
DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation.
Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.

Positive control substance(s):

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

Results and discussion

Positive control results:

Positive control (25 % (w/v) HCA in 1% Pluronic): Stimulation Index = 5.8

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.3

Test group / Remarks:

Test item at 25 (w/v) % in 1% Pluronic

Results 2

Parameter:

SI

Value:

0.7

Test group / Remarks:

Test item at 10 (w/v) % in 1% Pluronic

Results 3

Parameter:

SI

Value:

0.9

Test group / Remarks:

Test item at 5 (w/v) % in 1% Pluronic

Results 4

Parameter:

SI

Value:

1

Test group / Remarks:

Negative control (1% Pluronic)

Cellular proliferation data / Observations:

CLINICAL OBSERVATION
No mortality or signs of systemic toxicity were observed during the main study. No test item precipitate was observed on the ears of the animals. There were no indications of any irritancy at the site of application on the experimental animals.

BODY WEIGHT MEASUREMENT
No treatment related effects were observed on the mean body weight changes in the main study. Marked body weight loss (>5% reduction of body weight) was observed for one animal of the negative control group, positive control group and 25% (w/v) dose group, for two animals of the 10 % (w/v) dose group. However there was no clear dose relationship and all changes were considered as individual variability.

EAR THICKNESS MEASUREMENTS
The ear thickness values and revealing ear punch weights were within the historical control range. There was no indication of local irritation by visual assessment.

PROLIFERATION ASSAY
The results of the proliferation assay are summarized in Table 1 below. The appearance of the lymph nodes was normal in the negative control group and in all test item treated dose groups. Larger than normal lymph nodes were observed for some animals in the positive control group.

Any other information on results incl. tables

Table 1: DPM, DPN and Stimulation Index Values for all Groups

Test group name	Measured DPM/group	DPM	Number of lymph nodes	DPN	Stimulation Index
Background (5 % (w/v) TCA)	31 29	-	-	-	-
Negative control (1% Pluronic)	3926	3896.0	8	487.0	1.0
Test item 25 (w/v) % in 1% Pluronic	4946	4916.0	8	614.5	1.3
Test item 10 (w/v) % in 1% Pluronic	2725	2695.0	8	336.9	0.7
Test item 5 (w/v) % in 1% Pluronic	3507	3477.0	8	434.6	0.9
Positive control (25 % (w/v) HCA in 1% Pluronic)	22615	22585.0	8	2823.1	5.8

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, under the conditions of the present assay, the test item, tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.

Executive summary:

A study was performed to determine the skin sensitisation potential of the test item following dermal exposure according to OECD TG 429 and EU Method B.42. Based on the results of a preliminary compatability test 1% Pluronics PE9200 was selected as the solvent.The highest achievable concentration was 50 %w/v. A preliminary irritation/toxicity test was performed in CBA/CaOlaHsd mice (2 animals/dose) using doses of 50 or 25 %w/v in 1% Pluronic. Based on the observation recorded in the preliminary test, 25 %w/v was selected as the top dose for the main test.

In the main assay, 20 female mice were allocated to five groups of 4 animals each. Three groups received the test item (formulated in 1% Pluronic) at 25 %w/v, 10 %w/v or 5 %w/v. The negative control group received the vehicle (1 % Pluronic) and the positive control group received 25 %w/v HCA (dissolved in 1 % Pluronic).

The test item solutions were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).

No mortality or signs of systemic toxicity were observed during the main study. No test item precipitate was observed on the ears of the animals. There were no indications of any irritancy at the site of application on the experimental animals. Marked body weight loss (≥5%) was observed in the 25 %w/v and 10 %w/v dose groups and in the negative and positive control groups. No treatment related effects were observed on the mean body weight changes in the main study.

The stimulation index values were 1.3, 0.7 and 0.9 at concentrations of 25 %w/v, 10 %w/v and 5 % w/v, respectively.

The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A lymphoproliferative response in line with historic positive control data was noted for the positive control chemical, this result confirmed the validity of the assay.

In conclusion, under the conditions of the present assay, the test item, tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.