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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 25, 2017 to July 18, 20 17
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
Qualifier:
according to
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Version / remarks:
OECD Guidelines for the Testing of Chemicals, No.442B, July 22, 2010, "Skin Sensitization: Local
Lymph Node Assay: BrdU-ELISA"
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Name: Tetrakis (2,6-dimethylphenyl) m-phenylene bisphosphate
Abbreviation: PX-200
CAS number: 139189-30-3
Lot number: SK-290501
Molecular formula: C38H40O8P2
Molecular weight: 686.67
Purity: 99.6%
The test substance was treated as 100% in purity.
Melting point: 95.0 °C
Appearance at room temperature: White powder
Stability: Stable in the storage conditions
Storage: The test substance was put into an air-tight shaded container and was stored at room temperature (acceptable temperature: 10 to 30 °C) in desiccator of the test substance storage room.
Handling precaution: In order to avoid inhalation and contact with skin and eyes, chemically resistant gloves, a mask, a head cap, safety glass and lab coat were put on.
Specific details on test material used for the study:
No further details specified in the study report.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
Twenty six female CBA/J mice (SPF, 7 weeks old) were obtained from Charles River Laboratories Japan Atsugi Breeding Center. The CBA/J is a substrain of CBA/JN which is recommended in OECD TG 442B, and our testing facility has the LLNA historical control data of CBA/J.
At the receipt, since all animals showed good health conditions, they were received, weighed and assigned animal receipt numbers. The animals were quarantined and acclimatized for six days after the receipt and then the body weights were measured. Since no abnormalities were observed in clinical signs, excretions or body weight changes of any animals, the quarantine was completed and the animals were subsequently acclimatized. Two days before the first application of pre-screen test, five animals (receipt number 1-5) were housed individually and used for pre-screen test. The remaining 21 animals were further acclimatized, and two days before the first sensitization of the main study, the animal were weighed and were allocated to five groups (four animals per group) by body weight-stratified randomization. Any individual body weights were confirmed to be within the range of mean body weight ±20% at the group allocation. The remaining animal was excluded from the study. At the initiation of application, the animals were 8 weeks old in the pre-screen test and 9 weeks old in the main study.
The animals were housed ten animals or less per cage before the group allocation and then one animal per cage in the pre-screen test and four animals per cage in the main study. Clinical conditions and excretions were observed once or more daily.
The animals were identified by painting on the tail with red ink before group allocation and in the pre-screen test, and by painting with other colors after group allocation in the main study. Cages were identified by individual cards and racks were identified by indications of the study number, sex and test groups.

HOUSING CONDITIONS
The animals were housed in the barrier-system animal rooms (quarantine room number 2 during the quarantine period and animal room number 5 after the quarantine period) which were maintained at 21 to 25 °C, relative humidity at 40 to 70%, 10 to 15 air changes per hour and photoperiod of 12 hr light per day (light on at 7:00 and off at 19:00).
The animals were housed in polycarbonate cages (265W x426D x 150H mm, TOKIWA KAGAKU KIKAI) with softwood woodflakes (Sun-Flakes, lot number 170131, Charles River Laboratories Japan) before the group allocation and in po1ycarbonate cages (210W x 320D x 130H mm, CLEA Japan) with the woodflakes after the group allocation. Cages with woodflakes were changed at the completion of quarantine, group allocation and before carrying the animals from the animal room to the autopsy room. Stainless cage tops, water bottles and racks were changed at the group allocation.
Animals had free access to a pelleted diet (MF., lot number 170222, Oriental Yeast) and 3 to 5 ppm chlorinated water via water bottles. The chlorinated water was prepared by adding sodium hypochlorite (Purelox, OYALOX) to Hita City supply. Diets, woodflakes and housing materials were autoclaved at 121 °C for 30 min before using.
The information of the contaminants in diet and woodflakes was obtained from supplier (analyzed in Eurofins) and the results were confirmed to meet the requirements in CERI Hita.
Contaminants in drinking water were analyzed twice a year, and the analyzed data before the animal receipt were confirmed to meet the requirements in the water regulations of the "Ordinance on drinking water quality standards" (Ordinance Numbers 101 of Ministry of Health, Labour and Welfare).

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
The test substance was dissolved in AOO, which is a recommended vehicle in the OECD TG 442B, at a concentration of 50.0 w/v%. In addition, the solution was visually stable at room temperature for four hours after preparation. Therefore, the AOO was selected as a vehicle.
No. of animals per dose:
Pre-screen test: 5 animals
Main study: 4 animals per dose group
Details on study design:
PRE-SCREEN TEST
Objective: The objective of the pre-screen test was to set the dosage for the main study.
Grouping: Five dose levels of the test substance were set as follow. Since the test substance was solid, the highest concentration was set at 50.0 w/v%. The lower concentrations were determined according to OECD TG 442B.
Preparation of vehicle: On the first application day, 10 mL of acetone and 2.5 mL of olive oil were mixed to prepare AOO.
Preparation of test substance formulation: On the first application day, 0.50009 g of the test substance was dissolved in AOO and filled up to 1 mL to make 50.0 w/v% solution. The lower concentration solutions were prepared by serial dilution method as follows. These solutions had been stored at the cold place (acceptable temperature: to 10°C) and used for 3 days.
Application: Twenty-five microliters of each solution was applied to the dorsum of each ear of the animals using a micropipette once a day for three consecution days.
Clinical observation: All the animals were observed more than once a day from the first application day (Day 1) to terminal day (Day 6). Erythema scoring was not performed since no erythema was observed.
Body weights: Body weights of all the animals were measured on Day 1 and Day 6 using an electric balance.
Ear thickness: Thicknesses of each ear were measured on Day 1 (before application), Day 3 and Day 6 using a digital caliper (Digimatic Caliper, Mitutoyo ). Thickness of each ear was measured duplicate on each day, and the mean values were calculated.
Handling of animals after examinations: All the animals used in the pre-screen test were euthanized by cervical dislocation on Day 6.
Handling of dead or moribund animals: No moribund state or death occurred.
Evaluation of result: Dosages which induce following signs are supposed to be excluded from the main study.
-excessive irritation (erythema score 2: 3 and/or the ear thickness increases 25% or more)
-severe systemic toxicity and/or 5% or more body weight decrease

MAIN STUDY
Dose setting
In the pre-screen test, no abnormal changes which suggested systemic toxicity or excessive irritation were noted in any animals. Therefore, the dosages of the main study were set at 50.0, 25.0 and 10.0 w/v%.
Grouping
The test substance, vehicle control (AOO) and positive control (25.0 w/v% HCA) groups were set in the main study. Four animals were used in each group.

Preparations of vehicle, test substance formulation, positive control substance solution and BrdU solution
Vehicle: On each sensitization day, 10 mL of acetone and 2.5 mL of olive oil were mixed to prepare AOO.
Test substance formulation: On each sensitization day, 0.500 g of test substance was dissolved in AOO and filled up to 1 mL to make 50.0 w/v% solution. The actual weights were 0.50033 g (Day 1), 0.50001 g (Day 2) and 0.50001 g (Day 3). The 25.0 and 10.0 w/v% solutions were prepared by the same method as pre-screen test.
Positive control substance solution: On the first sensitization day, 0.25020 g of HCA was dissolved in AOO 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 three air-tight containers and the containers were shaded and preserved in refrigerator (acceptable temperature: 1-10 °C).
BrdU solution: Two days before the administration, 0.20002 g of 5-bromo-2'-deoxyuridine (BrdU, lot number M5H0079, Nacalai Tesque) was dissolved in physiological saline (lot number M6D75, Otsuka Pharmaceutical Factory) by ultrasonication and filled up to 20 mL to make 10 mg/mL solution. The BrdU solution was filtrated by a sterilizing filter (DISMIC®-25AS, pore size: 0.20 11m, lot number: 510221CD, Toyo roshi kaisha) and was stored in a sterile container in a refrigerator (acceptable temperature: 1-10 °C) until administration. The preparation was conducted under a yellow light condition, and the sterilization and after procedures were conducted in a clean bench.

Sensitization
The vehicle, test substance formulations and positive control solution were applied in the same way as the pre-screen test.

BrdU administration
Approximately 48 hours after the final sensitization, 0.5 mL ofBrdU solution was administrated intraperitoneally to each animal using a syringe and a needle (Terumo).

Clinical observations
Clinical observations were performed in the same way as the pre-screen test except for the erythema scoring.

Body weights measurements
Body weights were measured in the same way as for pre-screen test. The mean and the standard deviation were calculated for each group on each measurement day.

Collection and weights measurement of auricular lymph nodes
Approximately 24 hours after the BrdU administration, the animals were euthanized by cervical dislocation and each auricular lymph node was taken. The lymph nodes were carefully dissected and trimmed of surrounding tissue and fat, and weighed both sides together. The mean value and the standard deviation of the lymph nodes weights were calculated for each group. The lymph nodes were stored individually in the biomedical freezer (acceptable temperature: -30 to -10 °C).

BrdU labelling index
The auricular lymph nodes were defrosted at room temperature, homogenized and suspended in physiological saline (lot number 6HOON, Otsuka Pharmaceutical Factory). This suspension was filtered by cell strainer (Becton, Dickinson and Company), and dispensed into 3 wells per animal of a 96 well microplate (Costar). The BrdU uptake quantity was measured by ELISA using a commercial kit (Cell Proliferation ELISA, BrdU colorimetric, lot number 17267000, Roche Diagnostics). Absorbance at 3 70 nm with a reference wavelength of 492 nm was measured using multidetection microplate reader (FLUOstar OPTIMA, BMG LABTECH). The mean value of the 3 wells was defined as BrdU labelling index.

Handling of dead or moribund animals
No death or moribund state occurred.

Evaluation of result
Calculation of stimulation index (SI)
The SI for individual animals of was calculated by dividing the BrdU labelling index for each animal by the mean value of BrdU labelling index for the vehicle control group. The SIs were rounded off to one decimal place, and mean SI of each group was calculated.
SI = BrdU labelling index for individual animal/Men value of BrdU labelling indices for 4 animals of the vehicle control group

Evaluation method
Test substance is regarded as a "sensitizer" when the SI of the test substance group is 2.0 or more, and is regarded as a "non-sensitizer" when the SI of the test substance group is less than 1.6. When the SI is between 1.6-1.9, dose-response relationship and statistical significance are considered to evaluate the sensitization potential of the test substance.
However in this study, the statistical analysis was not conducted since the Sis of the any test substance groups were less than 1.6.

Validity of study
The test is regarded as valid when the SI of the positive control group is 1.6 or more.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The SI of the positive control group was 2.6.
The mean lymph node weights of the positive control group were 9.8 mg.
The mean BrdU labelling indices of the positive control group was 0.507.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
>= 0.9 - <= 1
Key result
Parameter:
other: BrdU labelling indices
Value:
> 0.168 - < 0.196
Cellular proliferation data / Observations:
The mean lymph nodes weights of the vehicle control group were 4.8 mg and those of 50.0, 25.0 and 10.0 w/v% test substance groups were 4.8, 4.7 and 5.8 mg, respectively.

Any other information on results incl. tables

Clinical signs in the pre-screen test

Exp. Group

Animal No.

Observation period

Group

Substance name

Concentration

(w/v %)

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Test substance

PX-200

2.50

1

-

-

-

-

-

-

5.00

2

-

-

-

-

-

-

10.0

3

-

-

-

-

-

-

25.0

4

-

-

-

-

-

-

50.0

5

-

-

-

-

-

-

-: no abnormalities detected

 

Body weights in the pre-screen test

Exp. Group

Animal No.

Body weight (g)

Group

Substance name

Concentration

(w/v %)

Day 1

Day 6a)

Test substance

PX-200

2.50

1

21.0

21.2     (101)

5.00

2

22.5

22.7     (101)

10.0

3

22.7

23.3     (103)

25.0

4

21.6

21.9     (101)

50.0

5

21.0

21.2     (101)

a)Figures in parentheses indicate percentages compare to the initial body weight (Day 1).

 

Thickness of auricle in the pre-screen test

Exp. Group

Animal No.

Thickness of auricle (mm)

Group

Substance name

Concentration

(w/v %)

Day 1

Day 3

Day 6

Left

Right

Average

Left

Right

Averagea)

Left

Right

Averagea)

Test substance

PX-200

2.50

1

0.18

0.19

0.19

0.17

0.18

0.18

(95)

0.16

0.18

0.17

(89)

5.00

2

0.18

0.17

0.18

0.17

0.18

0.18

(100)

0.17

0.19

0.18

(100)

10.0

3

0.15

0.18

0.17

0.16

0.17

0.17

(100)

0.17

0.18

0.18

(106)

25.0

4

0.14

0.17

0.16

0.16

0.17

0.17

(106)

0.17

0.16

0.17

(106)

50.0

5

0.16

0.17

0.17

0.16

0.17

0.17

(100)

0.18

0.17

0.18

(106)

a)Figures in parentheses indicate percentages compare to the initial thickness (Day 1).

 

Clinical signs in the main study

Exp. Group

Animal No.

Observation period

Group

Substance name

Concentration

(w/v %)

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Vehicle control

AOO

 

1

-

-

-

-

-

-

2

-

-

-

-

-

-

3

-

-

-

-

-

-

4

-

-

-

-

-

-

Positive control

HCA

25.0

5

-

-

-

-

-

-

6

-

-

-

-

-

-

7

-

-

-

-

-

-

8

-

-

-

-

-

-

Test substance

PX-200

10.0

9

-

-

-

-

-

-

10

-

-

-

-

-

-

11

-

-

-

-

-

-

12

-

-

-

-

-

-

25.0

13

-

-

-

-

-

-

14

-

-

-

-

-

-

15

-

-

-

-

-

-

16

-

-

-

-

-

-

50.0

17

-

-

-

-

-

-

18

-

-

-

-

-

-

19

-

-

-

-

-

-

20

-

-

-

-

-

-

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

HCA: α-hexylcinnamaldehyde

-: no abnormalities detected

 

Body weights in the main study

Exp. Group

Animal No.

Body weights (g)

Group

Substance name

Concentration

(w/v %)

Day 1

Day 6

Individual

Mean ± S.D.

Individual

Mean ± S.D.

Vehicle control

AOO

 

1

21.2

21.3 ± 0.9

22.8

22.2 ± 0.9

2

22.3

22.5

3

20.2

20.8

4

21.4

22.6

Positive control

HCA

25.0

5

21.2

21.2 ± 1.0

20.9

21.6 ± 1.8

6

21.1

21.5

7

20.0

19.8

8

22.4

24.1

Test substance

PX-200

10.0

9

20.5

21.0 ± 1.6

21.1

21.7 ± 1.3

10

23.0

23.2

11

19.1

20.3

12

21.3

22.2

25.0

13

21.7

21.4 ± 1.1

21.6

22.1 ± 1.0

14

22.7

23.4

15

20.1

21.2

16

21.0

22.0

50.0

17

22.6

21.8 ± 1.0

22.3

21.3 ± 1.0

18

22.3

21.9

19

20.3

20.0

20

21.8

21.0

S.D.: standard deviation

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

HCA: α-hexylcinnamaldehyde

 

Lymph node weights in the main study

Exp. Group

Animal No.

Lymph node weights

(mg)

Group

Substance name

Concentration

(w/v %)

Individual

Mean ± S.D.

Vehicle control

AOO

 

1

4.8

4.8 ± 0.4

2

5.1

3

5.0

4

4.2

Positive control

HCA

25.0

5

9.1

9.8 ± 0.7

6

9.3

7

10.3

8

10.5

Test substance

PX-200

10.0

9

6.3

5.8 ± 0.6

10

5.7

11

5.0

12

6.2

25.0

13

3.5

4.7 ± 1.3

14

4.7

15

4.2

16

6.5

50.0

17

4.8

4.8 ± 0.3

18

5.1

19

4.9

20

4.5

S.D.: standard deviation

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

HCA: α-hexylcinnamaldehye

 

BrdU labelling indices and stimulation indices in the main study

Exp. Group

Animal No.

BrdU labelling indices

Simulation indices

Group

Substance name

Concentration

(w/v %)

Individual

Mean ± S.E.

Individual

Mean ± S.E.

Vehicle control

AOO

 

1

0.152

0.194 ± 0.024

0.8

1.0 ± 0.1

2

0.247

1.3

3

0.155

0.8

4

0.222

1.1

Positive control

HCA

25.0

5

0.613

0.507 ± 0.052

3.2

2.6 ± 0.3

6

0.425

2.2

7

0.577

3.0

8

0.411

2.1

Test substance

PX-200

10.0

9

0.174

0.196 ± 0.018

0.9

1.0 ± 0.1

10

0.234

1.2

11

0.216

1.1

12

0.159

0.8

25.0

13

0.187

0.192 ± 0.019

1.0

1.0 ± 0.1

14

0.189

1.0

15

0.151

0.8

16

0.241

1.2

50.0

17

0.156

0.168 ± 0.004

0.8

0.9 ± 0.1

18

0.168

0.9

19

0.169

0.9

20

0.77

0.9

S.E.: standard error

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

HCA: α-hexylcinnamaldehyde

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In the main study, no abnormal findings which suggested excessive irritation or severe systemic toxicity were observed in clinical signs or body weight changes in any treatment groups. Therefore, all the data obtained were used for the assessment of skin sensitization potential. As a result, the Sis of the 50.0, 25.0 and 10.0 w/v% were 0.9, 1.0 and 1.0, respectively: the Sis of any dose levels were less than 1.6.
Therefore, under the conditions tested, PX-200 was considered to be a non-sensitizer. As for the positive control group, the SI was 2.6 and thus the study was confirmed to be valid.
Executive summary:

Local lymph node assay: BrdU-ELISA was performed in accordance with OECD TG 442B, using female CBA/J mice (SPF), and the skin sensitization potential of PX-200 was assessed by stimulation index (SI).

 

In the pre-screen test, 50.0, 25.0, 10.0, 5.00 and 2.50 w/v% test substance solutions, prepared with acetone: olive oil ( 4:1 v/v, AOO), were applied to mice daily for three consecutive days (one animal per dose level), and clinical observations, body weights measurements and ear thickness measurements were conducted. As a result, no abnormal changes which suggested excessive irritation or systemic toxicity were detected. Thus, the dose levels at 50.0, 25.0 and 10.0 w/v % were set for the main study.

 

In the main study, in addition to three test substance groups, vehicle control group which was applied with AOO and positive control group which was applied with a-hexylcinnamaldehyde (HCA) were set.

Four animals each were applied the vehicle, 25.0 w/v % HCA solution or test substance formulations daily for three consecutive days, and clinical observations and body weights measurements were performed. Approximately 48 hours after the final sensitization, 5-bromo-2'deoxyuridine (BrdU) was administered. Approximately 24 hours later, auricular lymph nodes were collected and their BrdU uptake quantities were measured to calculate the SIs. During the main study, no abnormal changes which suggested excessive irritation or systemic toxicity were noted. Therefore, all the data obtained were subjected to the skin sensitization evaluation.

 

Consequently, the SIs of the 50.0, 25.0 and 10.0 w/v % were 0.9, 1.0 and 1.0, respectively: the SIs of any dose levels were lower than 1.6. Therefore, under the conditions tested, PX-200 was considered to be a non-sensitizer.