Lithium phosphorodifluoridate

Endpoint summary

Description of key information

In an GLP-compliant, in vivo skin sensitisation study (Local Lymph Node Assay) conducted in accordance with OECD guidelines, the test substance was not sensitising to the skin.

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

Guideline:

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

Version / remarks:

22 July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31/05/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: ELEVAGE JANVIER, Route des Chènes Secs B.P. 4105 53940 LE GENEST-ST-ISLE, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks old (within one week)
- Weight at study initiation: 20.0 - 22.2 g
- Housing: Group caging / mice were provided with glass tunnel-tubes, Cage type: Type II. polypropylene / polycarbonate, Bedding was available to animals during the study (wood chips)
- Diet (e.g. ad libitum): Animals received ssniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm autoclavable complete diet for rats and mice" – breeding and maintenance, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 20 days
- Indication of any skin lesions: None. Only healthy animals were used for the study. Health status was certified by the veterinarian.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30-70%
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12h dark, 12h light

Vehicle:

other: 1% Pluronic

Concentration:

Test substance (Main study): 1, 2.5, 5 % w/v

No. of animals per dose:

4 animal per dose

Details on study design:

PRE-SCREEN TESTS:
The Preliminary Irritation/Toxicity Test I on CBA/J Rj mice using two doses (2 animals/dose) at test item concentrations of 50 and 25 (w/v) % in 1% Pluronic. Because of the observed mortality and excessive toxicity, an additional test was performed (Preliminary Irritation/Toxicity Test II) using three additional doses (2 animals/dose) at test item concentrations of 10, 5 and 2.5 (w/v) % in 1% Pluronic. The preliminary experiments were conducted in a similar experimental manner to the main study, but they were terminated on Day 6 and the radioactive proliferation assay was not performed.

The maximum concentration of test item in an acceptable solvent was established according to OECD guideline 429. Based on the observation of the solubility test, the maximum available concentration was 50 (w/v) %.

In the Preliminary Irritation / Toxicity Test, all mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema and scored using Table 2 [3]. Ear thickness was also 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.

During the Preliminary Irritation / Toxicity Tests, mortality and systemic toxicity was observed. One animal in the 50 (w/v) % dose group showed decreased activity, incoordination, tremors, weakness and was found moribund and euthanized on Day 1. The other animal in this group showed decreased activity, tremors and incoordination. This animal died on Day 2. Animals of the 25 (w/v) % dose group showed signs of systemic toxicity on Day 2. They were found moribund and euthanized on Day 2. Animals of 10 (w/v) % dose group showed intermittent tremors on Days 4-6 and hunched back on Days 5-6. Additionally, marked body weight loss (20.6%) was observed on the animals in this group. Animals in the 5 and 2.5 (w/v) % dose group were symptom-free during the preliminary experiment.

Ear thickness of the animals was measured using by a thickness gauge on Days 1, 3 and 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals. The ear punch weights of the surviving animals were in the historical control range.

The draining auricular lymph nodes of the animals were visually examined: smaller than normal lymph nodes were observed in the 10 (w/v) % dose group, larger than normal lymph nodes were observed in the 5 (w/v) % group and the nodes were considered to be normal in the 2.5 (w/v) % dose group (subjective judgement by analogy with observations of former experimental).

Based on these observations, the 50, 25 and 10 (w/v) % dose groups exceeded the maximum toxicity criteria. Therefore, the 5 and 2.5 (w/v) % doses were acceptable for the main test.

MAIN STUDY
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. 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.

ANIMAL ASSIGNMENT AND TREATMENT
The animals were randomised and allocated to the experimental groups. The randomisation was checked by computer software using the body weight to verify the homogeneity and variability between the groups.

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.

Positive control substance(s):

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

Statistics:

None

Positive control results:

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. The positive control substance was examined at a concentration of 25 % in the relevant vehicle (1% Pluronic) using CBA/J Rj mice.

No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A significant lymphoproliferative response (stimulation index value of 3.1) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.

Furthermore, the DPN values observed for the vehicle and positive control substance in this experiment were within the historical control range. Each treated and control group included 4 animals.

Parameter:

SI

Value:

1

Test group / Remarks:

Negative control

Parameter:

SI

Value:

0.9

Test group / Remarks:

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

Parameter:

SI

Value:

1.1

Test group / Remarks:

Test item: 2.5 (w/v) % in 1% Pluronic acid

Parameter:

SI

Value:

1.4

Test group / Remarks:

Test item: 1 (w/v) % in 1% Pluronic

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
See table below

DETAILS ON STIMULATION INDEX CALCULATION
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.

EC3 CALCULATION
Not applicable.

CLINICAL OBSERVATIONS:
No mortality or signs of systemic toxicity were observed during the study. There were no indications of any irritancy at the site of application.

BODY WEIGHTS
No treatment related effects were observed on animal body weights, however marked body weight loss (7.4 %) was detected for one animal in the 2.5 (w/v) % dose group.

DPM, DPN and Stimulation Index Values for all groups

Test Group	Measured DPM/group	DPM	No. of lymph nodes	DPN	Stimulation Index
Background (5% (w/v)% TCA)	34.0	-	-	-	-
Negative (vehicle) control (1% Pluronic acid)	1373	1339.0	8	167.4	1.0
Test item 5% (w/v) in 1% Pluronic	1226	1192.0	8	149.0	0.9
Test item 2.5% (w/v) in 1% Pluronic	1562	1528.0	8	191.0	1.1
Test item 1% (w/v) in 1% Pluronic	1878	1844.0	8	230.5	1.4
Positive control 25 (w/v) % HCA in 1% Pluronic acid	4194	4160.0	8	520.0	3.1

Interpretation of results:

GHS criteria not met

Conclusions:

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the data available, the substance does not fulfil the criteria to be classified as skin senitiser in accordance with the CLP Regulation (EC1272/2008) and therefore is not classified for this endpoint.