3,9-dibenzyl-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane 3,9-dioxide

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Test material information

Constituent 1

Reference substance name:

3,9-dibenzyl-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane 3,9-dioxide

EC Number:

243-869-3

EC Name:

3,9-dibenzyl-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane 3,9-dioxide

Cas Number:

20544-37-0

Molecular formula:

C19H22O6P2

IUPAC Name:

3,9-dibenzyl-2,4,8,10-tetraoxa-3λ⁵,9λ⁵-diphosphaspiro[5.5]undecane-3,9-dione

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot No. of test material: 44216018
- Purity test date: 99.9%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test substance was put into an air-tight brown container and stored in the test substance storage room at room temperature (acceptable range: from 10°C to 30°C).
- Stability under test conditions: Stable against water and heat.
- Solubility and stability of the test substance in the solvent/vehicle:
Solubility to solvent
Solvent Solubility Stability
Acetone 700 mg/L Stable
Dimethyl sulfoxide 3.6E+04 mg/L Stable

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Japan, Inc., Atsugi Breeding Center
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals: SPF
- Age at study initiation: 7 weeks old
- Weight at study initiation: 18.9 - 23.8 g
- Housing: The animals were housed 10 animals or less per cage before the group allocation, 1 animal per cage for the pre-screen test and 4 animals per cage for the main study. The animals were housed in barrier-system animal rooms in polycarbonate cages (265W ×426D ×150H mm) with wood chips (Sun-Flakes, Lot number 181120, Charles River Laboratories, Japan, Inc.) before the group allocation and in polycarbonate cages (210W × 320D × 130H mm) with the wood chips after the group allocation. Cages with wood chips were changed at the completion of quarantine, at group allocation and before carrying the animals from animal room to the autopsy room. Stainless cage top, water bottle and rack were changed at the group allocation.
- Diet (e.g. ad libitum): Animals had free access to a pelleted diet (MF, Lot number 181224, Oriental Yeast)
- Water (e.g. ad libitum): Animals had free access to 5 ppm chlorinated water via water bottles
- Acclimation period: for 6 days after the receipt

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 25
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

The test substance was suspended homogenously in DMF at a concentration of 50.0 w/v%.

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The test substance was suspended homogenously in DMF at a concentration of 50.0 w/v%, but inhomogeneous in AOO, MEK or DMSO. Twenty hours later, the test substance settled at the bottom of DMF although easily re-suspended by agitation. Therefore DMF was selected as a vehicle.
- Irritation: none
- Systemic toxicity: none
- Ear thickness measurements: Thickness of each ear was measured on Day 1 (before application), Day 3 and Day 6 using a resin vernier caliper (digimatic caliper, Mitutoyo). Thickness of each ear was measured duplicate on each day, and the mean values were calculated.
- Erythema scores: the Erythema was not detected.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay: BrdU-ELISA
- Criteria used to consider a positive response: The individual SIs of all the animals were calculated by dividing the BrdU labelling index for each animal by the mean BrdU labelling index for the vehicle control. Individual SIs were rounded off to one decimal place. Also mean SI and standard error of each group were calculated. Test substance was regarded as a “sensitiser” when the SI of the test substance group was 2.0 or more, and was regarded as “non-sensitiser” when the SI of the test substance group was less than 1.6. When the SI was between 1.6 and 1.9, dose-response relationship and statistical significance were supposed to be considered.

TREATMENT PREPARATION AND ADMINISTRATION:
- Preparations of test substance formulation, positive control substance solution and BrdU solution:
1) Test substance formulation: On each sensitisation day 0.500 g of the test substance was suspended in DMF and filled up to 1 mL to make a 50.0 w/v% formulation. The actual weights of the test substance were 0.50025 g (Day 1), 0.50023 g (Day 2) and 0.50014 g (Day 3). Then 25.0 and 10.0 w/v% suspensions were prepared.
2) Positive control substance solution: On the first sensitisation day, 0.25029 g of HCA was dissolved in DMF and filled up to 1 mL to make a 25.0 w/v% HCA solution under a yellow light condition. The solution was subdivided into 3 air-tight containers and the containers were shaded and preserved in a refrigerator (acceptable temperature: 1-10ºC).
3) BrdU solution: Two days before the administration, 0.50018 g of 5-bromo-2’-deoxyuridine (BrdU, Lot number M8F5463, Nacalai Tesque) was dissolved in physiological saline (Lot number K8H70, Otsuka Pharmaceutical Factory) by ultrasonication and filled up to 50 mL to make a 10 mg/mL solution. The BrdU solution was filtrated by a sterilizing filter (DISMIC®-25AS, pore size: 0.20 μm, Toyo roshi kaisha) and was stored in a sterile container in a refrigerator (acceptable temperature: 1-10ºC) until administration. The preparation was conducted under a yellow light condition, and the sterilization and after procedures were conducted in a tabletop clean booth.
- Application: Twenty-five μL of each formulation was applied to the dorsum of each ear of the animals using a micropipette once a day for 3 consecutive days.
- Clinical observation: All the animals were observed once or more daily from the first application day (Day 1) to terminal day (Day 6).
- Ear thickness: Thickness of each ear was measured on Day 1 (before application), Day 3 and Day 6 using a resin vernier caliper (digimatic caliper, Mitutoyo). Thickness of each ear was measured duplicate on each day, and the mean values were calculated.
- Treatment of animals after examinations: All the animals used in the pre-screen test were humanely killed by cervical dislocation on Day 6.
- Treatment of dead or moribund animals: No dead or moribund animals were observed.
- BrdU administration: Approximately 48 hours after the final sensitisation, 0.5 mL of BrdU solution was administrated intraperitoneally to each animal using a syringe and a needle (Terumo).
- BrdU labelling index: The auricular lymph nodes were defrosted at room temperature, homogenized and suspended in physiological saline (Lot number 8H76N, Otsuka Pharmaceutical Factory). This suspension was filtered by cell strainer and dispensed into 3 wells per animal of a 96 well microplate (Costar). The BrdU uptake levels were measured by ELISA using a commercial kit (Cell Proliferation ELISA, BrdU colorimetric, lot number 34570800, Roche Diagnostics). Absorbance at 370 nm with a reference wavelength of 492 nm was measured using multidetection microplate reader (FLUOstar Omega, BMG LABTECH). The mean value of the 3 wells was defined as BrdU labelling index.

Positive control substance(s):

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

Results and discussion

Positive control results:

The mean lymph node weights for the positive control group was 10.2 mg. This results in a SI for the positive control group of 3.33.
The test was regarded as valid as the SI of the positive control group was more than 1.6.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.98

Test group / Remarks:

1

Results 2

Key result

Parameter:

SI

Value:

1.05

Test group / Remarks:

2

Results 3

Key result

Parameter:

SI

Value:

0.95

Test group / Remarks:

3

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA : The auricular lymph nodes were defrosted at room temperature, homogenized and suspended in physiological saline (Lot number 8H76N, Otsuka Pharmaceutical Factory). This suspension was filtered by cell strainer and dispensed into 3 wells per animal of a 96 well microplate (Costar). The BrdU uptake levels were measured by ELISA using a commercial kit (Cell Proliferation ELISA, BrdU colorimetric, lot number 34570800, Roche Diagnostics). Absorbance at 370 nm with a reference wavelength of 492 nm was measured using multidetection microplate reader (FLUOstar Omega, BMG LABTECH). The mean value of the 3 wells was defined as BrdU labelling index.

DETAILS ON STIMULATION INDEX CALCULATION : The individual SIs of all the animals were calculated by dividing the BrdU labelling index for each animal by the mean BrdU labelling index for the vehicle control. Individual SIs were rounded off to one decimal place. Also mean SI and standard error of each group were calculated.
Test substance was regarded as a “sensitiser” when the SI of the test substance group was 2.0 or more, and was regarded as “non-sensitiser” when the SI of the test substance group was less than 1.6. When the SI was between 1.6 and 1.9, dose-response relationship and statistical significance were supposed to be considered.

CLINICAL OBSERVATIONS: No abnormalities were noted in any group.

BODY WEIGHTS: No abnormal changes were noted in any treatment group compared to those of vehicle control group.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

No abnormal findings which suggested excessive irritation or severe systemic toxicity were observed in clinical signs or body weight changes in any treatment group.
As a result, the SIs for the 50.0, 25.0 and 10.0 w/v % test substance groups were 0.98, 1.05 and 0.95: all the SIs were less than a cut-off value of 1.6.
Therefore, under the conditions tested, 3,9-dibenzyl-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane 3,9-dioxide was considered to be a non-sensitiser.

Executive summary:

Local lymph node assay: BrdU-ELISA was performed in accordance with the OECD TG 442B using female CBA/J mice, and the skin sensitisation potential of 3,9-dibenzyl-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane 3,9-dioxide was assessed by calculating the stimulation index (SI).

In the pre-screen test, 50.0, 25.0 and 10.0 w/v % of test substance formulations, prepared with N,N-dimethylformamide (DMF), were applied to the dorsum of both ears of one animal/dose level once daily for 3 consecutive days. Clinical observations, body weight measurements and ear thickness measurements were performed. As a result, no abnormal changes which suggested excessive irritation or systemic toxicity were detected. Thus, the dose levels of the main study were set at 50.0, 25.0 and 10.0 w/v%.

In the main study, in addition to the test substance groups, vehicle control group which was applied with DMF and positive control group which was applied with 25.0 w/v % α-hexylcinnamaldehyde (HCA) were set. The vehicle, HCA and test substance formulations were applied to the dorsum of both ears of 4 animals/group once daily for 3 consecutive days. Clinical observations and body weight measurements were performed. Approximately 48 hours after the final sensitisation, 5-bromo-2’deoxyuridine (BrdU) was administered. Approximately 24 hours later, auricular lymph nodes were collected, the BrdU uptake levels were measured by ELISA, and the SIs were calculated. During the main study, no abnormal changes which suggested excessive irritation or systemic toxicity were noted. Therefore, all the data obtained were used to the skin sensitisation evaluation.

As a result, the SIs of the 50.0, 25.0 and 10.0 w/v% test substance groups were 0.98, 1.05 and 0.95: all the SIs were less than a cut-off value of 1.6.Therefore, under the conditions tested, 3,9-dibenzyl-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane 3,9-dioxide was considered to be a non-sensitiser.