Biphenyl-4,4'-diyl tetrakis(2,6-dimethylphenyl) bis(phosphate)

Endpoint summary

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

Study period:

19-25 January 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Data have been generated according to current internationally recognised study guidelines and in accordance with GLP

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Species and strain: CBA/J Rj mice
Source: ELEVAGE JANVIER Route des Chènes Secs B.P. 4105 53940 LE GENEST-ST-ISLE, France
Hygienic level at arrival: SPF
Hygienic level during the study: Standard housing conditions
Justification of strain: On the basis of OECD Guideline, mice of CBA/Ca or CBA/J strain can be used. Females are used because the existing database is predominantly based on females.
Number of animals: 4 animals / treatment group
Sex: female, nulliparous, non pregnant Age of animals at starting: 9 – 10 weeks old Body weight range at starting: 21.1 – 23.4 grams (The weight variation in animals involved in the study did not exceed ± 20 % of the mean weight)
Acclimatization time: 13 days

Animal health: Only healthy animals were used for the study. Health status was certified by the veterinarian.
Housing / Enrichment: Group caging / mice were provided with glass tunnel-tubes
Cage type: Type II. polypropylene/ polycarbonate Bedding: Bedding was available to animals during the study Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 - 70 % *
Ventilation: 15-20 air exchange/hour
The temperature and relative humidity were recorded twice every day during the acclimatisation and experimental phases.
Room/Cabinet (non-radioactive phase): 244/7
Room/Cabinet (radioactive phase): 139 – 140
*: Minor variations from the target humidity ranges were observed during acclimatisation period (Room/Cabinet: 244/2). These deviations were considered to have no impact on the animal health, as certified by the Clinical Veterinarian, or on the outcome of the study and interpretation of the results due to their low magnitude.

Animals received ssniff SM R/M-Z+H "Autoclavable complete diet for rats and mice – breeding and maintenance" (Batch number: 802 4830 Expiry Date: May 2011) produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), ad libitum.
Animals received tap water from the municipal supply from 500 ml bottle, ad libitum.
Lignocel Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (D-73494 Rosenberger (Germany) Holzmühle 1) was available to animals during the study.
A unique number written on the tail with a permanent marker identified each animal.

Vehicle:

propylene glycol

Concentration:

50, 25 and 10 (w/v) %,

No. of animals per dose:

4

Details on study design:

A Preliminary Irritation/Toxicity Test was performed on CBA/J Rj mice using two doses, at test item concentrations of 50 and 25 (w/v) %, respectively. This preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 with a body weight measurement and radioactive proliferation assay was not performed. An assessment of ear swelling was made by measuring thickness on days 1, 3 and 6, and the weight of an ear punch on day 6.

During the assay each mouse was topically dosed on the dorsal surface of each ear with 25 μl of the appropriate formulation applied using a pipette. 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.

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 25G1" hypodermic needle with 1 ml sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).

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.

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.

After the final wash, supernatant were removed leaving a small volume (<0.5 ml) of supernatant above each pellet. Each pellet was gently agitated before suspending the LNCs in 3 ml of 5% TCA (trichloroacetic acid) for precipitation of macromolecules. After incubation with 5% TCA at 2-8 °C overnight (approximately 18 hours) precipitate was recovered by centrifugation at 190 x g for 10 minutes, supernatants were removed and pellets were suspended in 1 ml of 5% TCA and dispersed using ultrasonic water bath. Each precipitate was transferred to a suitable sized scintillation vial with 10 ml of scintillation liquid and thoroughly mixed. The vials were loaded to a β-scintillation counter and 3HTdR incorporation was measured for up to 10 minutes per sample. The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Similarly, background 3HTdR levels were also measured in two 1 ml aliquots of 5% TCA.

Positive control substance(s):

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

Positive control results:

DPN: 1944.3
Stimulation Index Values: 27.9

Parameter:

SI

Remarks on result:

other: 50 % in PG: 0.9 25 % in PG: 1.5 10 % in PG: 0.9 Negative control: 1.0

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: 50 % in PG: 63.9 25 % in PG: 101.5 10 % in PG: 65.9 Negative control: 69.6

No mortality or systemic clinical signs were observed during the study. No treatment related effects were observed on animal body weights in any treated groups.

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of the test the substance displays no sensitising potential.

Executive summary:

The skin sensitisation potential of the substance was measured following exposure to mice according to the LLNA test as prescribed in the OECD 429 test method in accordance with GLP. Under the conditions of the test the substance displays no sensitising potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Based on the results of the Preliminary Compatibility Test and on the recommendations of the OECD Guideline, the test item was suspended in Propylene Glycol (PG). The achievable maximum concentration was 50 (w/v) %.

The Preliminary Irritation/Toxicity Test was performed in CBA/J Rj mice using two doses (test item concentrations of 50 and 25 (w/v) %) in the selected vehicle. The applicability and biocompatibility of the test item on the ears of animals at the maximum concentration of test item of 50 (w/v) % was acceptable.

In the main assay, twenty female CBA/J Rj mice were allocated to five groups of four animals each:

- three groups received the appropriate formulation of PX-202 at concentrations of 50, 25 and 10 (w/v) %,

- the negative control group received PG,

- the positive control group received 25 % α-Hexylcinnamaldehyde (HCA) in PG.

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 systemic clinical signs were observed during the study.

No treatment related effects were observed on animal body weights in any treated groups.

Stimulation index values of the test item were 0.9, 1.5 and 0.9 at treatment concentrations of 50 %, 25 % and 10 (w/v) %, respectively.

α-Hexylcinnamaldehyde (25 (w/v) % dissolved in PG) was used as a positive control to demonstrate the appropriate performance of the assay. A significant lymphoproliferative response (stimulation index value of 27.9) was noted for the positive control chemical and this result confirmed the validity of the assay.

Justification for selection of skin sensitisation endpoint:
Data have been generated according to current internationally recognised study guidelines and in accordance with GLP

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The skin sensitisation potential of the substance was measured following exposure to mice according to the LLNA test as prescribed in the OECD 429 test method in accordance with GLP. Under the conditions of the test the substance displays no sensitising potential.