tert-butylphenyldiphenyl phosphate (tBuTPP)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

22 December 2009 - 13 January 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted according to OECD guideline 429 and under GLP conditions.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Remarks:

Statement of Compliance

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of t-butylphenyldiphenyl phosphate and bis-t-butylphenylphenyl phosphate and triphenyl phosphate

IUPAC Name:

Reaction mass of t-butylphenyldiphenyl phosphate and bis-t-butylphenylphenyl phosphate and triphenyl phosphate

Constituent 2

Reference substance name:

Phosflex 71B

IUPAC Name:

Phosflex 71B

Details on test material:

- Name of test material (as cited in study report): Phosflex 71B
- Physical state: Liquid
- Lot/batch No.: confidential
- Expiration date of the lot/batch: confidential
- Stability under test conditions: stable
- Storage condition of test material: at room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Limited, Bicester, Oxon, UK
- Age at study initiation: eight to twelve weeks old
- Weight at study initiation: 15 to 23 g
- Housing: individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet: ad libitum Global Rodent diet
- Water: ad libitum mains tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: no data

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

0% (vehicle), 25%, 50%, and 100%

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Concentration: 100 % - undiluted test material (maximum concentration in accordance with test guideline).
- Irritation: No signs of systemic toxicity were noted.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response: Exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index (SI).

TREATMENT PREPARATION AND ADMINISTRATION: The test material was used undiluted and freshly prepared as a solution in acetone/olive oil 4:1. The test material was formulated within 2 hours of it being applied to the test system.
The mice were treated by daily application of 25 ul of the appropriate concentration of the test material to the dorsal surface of each ear for 3 consecutive days (Days 1, 2, 3). The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette (proliferative capacity was determined by incorporation of 20 uCi 3H-methyl thymidine per mouse).

Positive control substance(s):

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

Statistics:

Data was processed to give group mean values for disintegrations per minute (DPM) and standard deviations where appropriate. Individual and group mean DPM values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett's Multiple Comparison test was used and for non-homogenous datasets Dunnett's T3 Multiple Comparison Method was used.

Results and discussion

Positive control results:

a-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index (SI) of greater than 3 (5.62) when tested at a concentration of 15% v!v in acetone/olive oil 4:1, and was thus considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: 25%: 3.36 50%: 4.00 100%: 3.24

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean dpm/Animal (Standard Deviation) Vehicle Control: 2649.69 (± 907 .65) Test Material: 25%: 8892.72 (± 4480.61) 50%: 10605.76 (±3461.49) 100%: 8587.76 (±6743.92)

Any other information on results incl. tables

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Bodyweight changes of the test animals between Day I and Day 6 were comparable to those observed in the corresponding control group animals over the same period, except for one vehicle control animal which showed a greater than expected bodyweight gain.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

Under the conditions of this study, Stimulation Indices (SI) of 3.36, 4.00, and 3.24 were calculated for 25%, 50%, and 100% Phosflex 71B, respectively. Considering the SI threshold value of 3 as stated in Annex I of 1272/2008/EC and Annex VI of 67/548/EEC, the substance needs to be classified as a sensitizer.

Executive summary:

A Local Lymph Node Assay (LLNA) was performed to assess the skin sensitisation potential of Phosflex 71B in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements of the OECD Guideline No. 429 (24 April 2002) and Method B42 of EC Regulation No. 440/2008. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100%, this concentration was selected as the highest dose investigated in the main test of the LLNA. Three groups, each of 5 animals, were treated with 50 ul (25 ul per ear) of the undiluted test material or the test material as a solution in acetone/olive oil 4:1 at concentrations of 50% or 25% v/v. A further group of 5 animals was treated with acetone/olive oil 4:1 alone. A concurrent positive control test, using a group of 5 animals, was also performed with the known sensitiser, α -Hexylcinnamaldehyde tech., 85%, at a concentration of 15% v/v in acetone/olive oil 4:1. Proliferative capacity was determined by incorporation of 20 uCi 3H-methyl thymidine per mouse and subsequent liquid scintillation counting. Mortality, clinical signs and body weight were recorded. No mortality or clinical signs were noted and body weights were within the range commonly recorded for the test animals. The mean amount of disintegration per minute (DPM) that were measured by liquid scintillation counting were: 8892.72, 10605.76, and 8587.76 for the concentrations of 25%, 50%, and 100%, respectively. Under the conditions of this study, Stimulation Indices (SI), expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group, were as follows: 25% Phosflex 71B: 3.36; 50% Phosflex 71B: 4.00; 100% Phosflex 71B: 3.24. α-Hexyicinnamaldehyde, tech., 85% gave a SI of 5.62. Considering the SI threshold value of 3 as stated in Annex I of 1272/2008/EC and Annex VI of 67/548/EEC, Phosflex 71B needs to be classified as a sensitizer.