Polysodium 4-amino-5-hydroxy-3-[{8-hydroxypolysulfonato-7-[{sulfonato-4-[(sulfonatophenyl)diazenyl]phenyl}diazenyl]-2-naphthyl}diazenyl]-6-[(4-nitro-sulfonatophenyl)diazenyl]naphthalenepolysulfonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 August 2013 to 07 October 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD guidelines in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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)

Test material

Test material information

Constituent 1

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

ANIMALSTwenty nine female CBA/J mice (SPF, 8 weeks old) were obtained from Charles River Japan Atsugi Breeding Center. CBA/J is substrain of CBA/JN which is recommended strain in OECD TG442B, and our test facility have the historical control data of CBA/J.The animals were acclimatized for 9 days including 7 days quarantine period. After 9 days of the receipt, 6 animals (receipt number: 1-6) were used for pre-screen test. The other 23 animals were subsequently acclimatized until 14 days after the receipt (two days before the first sensitization in the main study). After the body weights measurements, the animals were allocated to five groups (four animals per group) by body weight-stratified randomization for the main study and then it was confirmed that any individual body weighed were within the range of mean body weight ±20%. Three remaining animals were excluded from the study.At the time of sensitization, animals were 9 weeks old in pre-screen test and 10 weeks old in the main study.The animals were housed 10 animals or less per cage during the quarantine/acclimatization periods, one animal per cage in the pre-screen test and 4 animals per cage in the main study.Clinical conditions and excretions were observed once or more a day until the first sensitization.The animals were identified by painting on the tail with red ink before grouping and by painting with other colors after grouping. Cages were identified by individual cards and racks were identified by indications of the study number.HOUSING CONDITIONSThe animals were housed in the barrier-system animal rooms (Quarantine Room No. 2 during the quarantine period and Animal Room No. 4 after the quarantine period) which were maintained at 21-25°C, relative humidity at 40-70%, 10-15 air changes per hour and photoperiod of 12 hr light per day (light on at 7:00 and off at 19:00). The actual temperature and relative humidity were 22.6-24.3 °C and 49.7-60.5%, respectively.The animals were housed in polycarbonate cages (265W x426D x 150H mm, TOKIWA KAGAKU KIKAI) with softwood woodflakes (Sun-Flakes, Lot No. 130212, Charles River Japan) before the grouping and in polycarbonate cages (21 OW x 320D x 130H mm, CLEA Japan, Inc.) with softwood woodflakes after the grouping. Cages and woodflakes were changed at the grouping and before carrying the animals from the animal room to the autopsy room. Water bottle and rack were changed at the grouping.Animals had free access to a pelleted diet (MF. Lot No. 130515, Oriental Yeast) and about 5 ppm chlorinated water via water bottles. The chlorinated water was prepared by adding sodium hypochlorite (Purelox, OYALOX Co., Ltd) to Hita City supply. Diets, woodflakes and housing materials were autoclaved before using at 121°C for 30 min.The information of the contaminants in diet and woodflakes were obtained from supplier (analyzed in Eurofins) and confirmed to meet the requirements in CERI Hita which referred to the "Toxic Substances Control Act of United States Environmental Protection Agency" (1979).Contaminants in drinking water were analyzed twice a year, and the analyzed data before the receipt of the animals were confirmed to meet the requirements in the water regulations of the "Ordinance on drinking water quality standards" [Ordinance No. 101 of Ministry of Health, Labour and Welfare].

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Reported in table form - see Any other information

No. of animals per dose:

Reported in table form - see Any other information

Details on study design:

PRE-SCREEN TESTGrouping: Six dose levels of the test substance were used – detailed in table form, see Any other information.Preparation of vehicle and the test substance formulation:-Vehicle (AOO): Acetone and olive oil were mixed at the ratio of 4:1 (v/v) on each sensitization day.-Test substance formulation: Approximately 0.3 g of the test substance was collected, grounded in mortar and then 0.250 g was weighed and suspended in 1 mL of AOO to make 25.0 w/v% formulation. The actual weights of grounded test substance on each sensitization day were 0.25023 g Day 1), 0.25000 g (Day 2) and 0.25030 g (Day 3). Lower concentrations were prepared by serial dilution – detailed in table form, see Any other information.Sensitization: Twenty-five microliter of each formulation was applied to the dorsum of each ear of the animals using a micropipette once a day for three days.Clinical observation: All animals were observed more than once a day from the first sensitization day (Day 1) to terminal day (Day 6). Scoring was not performed since erythema was not observed.Body weights: Body weights of all animals were measured on Day 1 and Day 6 using an electric balance (SARTORIUS).Ear thickness: Thicknesses of each ear were measured on Day 1 (before sensitization), approximately 48 hours after the first sensitization and Day 6 using a resin vernier caliper (digimatic caliper, Mitutoyo). Thickness of each ear was measured twice on each day, and the mean values were calculated.Handling of the animals after examinations: All animals used in the pre-screen test were euthanized by cervical dislocation on Day 6.Evaluation of the result: Dosages which induce following signs were excluded from the main study.-excessive irritation (Erythema score ≥ 3 or the ear thickness on Day 6 increases 25% and more than those on Day 1)-severe systemic toxicityMAIN TESTDose setting: In the pre-screen test, systemic toxicity or excessive initation were not observed in any groups. Therefore, the doses were set at 25, 10 and I% in the main study. The low dose was set as 1% to classify the test substance into the hazard class of the Globally Harmonized System of Classification and Labeling of Chemicals (GHS).Grouping: The test substance, vehicle control (AOO) and positive control (25.0% HCA) groups were set in the main study. Four animals were used in each group – detailed in table form, see Any other informationPreparations of the vehicle, test substance formulation, positive control substance solution and BrdU solution:Vehicle: Acetone and olive oil were mixed at the ratio of 4:1 (v/v) on each sensitization day.Test substance formulation: Approximately 0.5 g of the test substance was collected and grounded in mortar. A portion was weighed and suspended in AOO as bellow to make appropriate concentrations. Each formulation was ultrasonicated to make homogenously, detailed in table form – see Any other informationPositive control substance solution: On the day before the first sensitization, 0.25015 g of HCA was dissolved in AOO and fill up to the volume of 1 mL to make a 25.0 w/v% HCA solution. The solution was subdivided into three shading air-tight container (glass) and preserved in refrigerator (tolerance temperature: 1-10 °C).BrdU solution: Two days before the administration, 0.20046 g of 5-bromo-2'-deoxyuridine (BrdU, LotNo. M1B6181, Nacalai Tesque) was dissolved in physiological saline (Lot No. K2H73, Otsuka Pharmaceutical Factory) by ultrasonication and fill up to 20 mL to make 10 mg/mL solution under yellow light. The BrdU solution was sterilized by a sterilizing filter (DISMIC®, pore size: 0.20 11m, Lot no.: 206251CD, ADVANTEC) and was stored in an airtight container in refrigerator (tolerance temperature: 1-1 0°C) until administration. The preparation was conducted under yellow light and the handling after the sterilization was conducted in the clean bench.Sensitization procedure: Each formulation or solution was applied in the same way as for the pre-screen test.BrdU administration: Approximately 48 hours after the final sensitization, 0.5 mL of BrdU 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 for the pre-screen test.Body weight measurements: Body weights were measured in the same way as for the pre-screen test. The mean and the standard deviation were calculated for each group on each measurement day.Collection and weights measurement of lymph nodes: Approximately 24 hours after BrdU administration, animals were euthanized by cervical dislocation and each auricular lymph node was taken. The lymph nodes were carefully dissected and trimmed of fascia and fat, and weighed both sides together. The mean values and the standard deviations of the lymph node weights were calculated for each group. The lymph nodes were stored individually in the biomedical freezer (temperature: -20°C,).BrdU labelling index: The BrdU was measured by ELISA using a commercial kit (Kit: Cell Proliferation ELISA, BrdU colorimetric, Lot No. 130719900, Roche Diagnostics). The auricular lymph nodes were defrosted to room temperature, homogenized and suspended in physiological salin (Lot No. 2F81N Otsuka Pharmaceutical Factory). This suspension was filtered by cell strainer, and dispensed into a 96 well microplate. Absorbance at 370 nm with a reference wavelength of 492 nm was measured using microplate reader (FLUOstar OPTIMA, BMG LABTECH). Each sample was analyzed in triplicate and the mean value was defined as BrdU labeling index.

Positive control substance(s):

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

Statistics:

The SI for test substance group and positive control group in individual animal was calculated by dividing the BrdU labeling index for each animal by the mean values of BrdU labeling index for the vehicle control. The mean and standard errors of SI were calculated for each group and rounded off to one decimal place.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The Sis of the BK-659 treatment groups were all less than 1.6.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: DPM not reported.

Any other information on results incl. tables

RESULTS

PRE-SCREEN TEST

Clinical observations: No abnormalities were observed in any groups.

Body weights: No abnormal changes were observed in any groups.

Ear thickness: The ear thicknesses on Day 3 and 6 were comparable to those on Day 1 and did not increase more than 25%.

MAIN STUDY

Clinical observations: No abnormalities were observed in any treatment or control groups.

Body weights: No abnormal changes were observed in any groups compare to that of vehicle control group.

Lymph node weights: The mean lymph nodes weight of the vehicle control group was 5.1 mg and those of 1, 10 and 25 %test substance groups were 4.7, 4.8 and 4.7 mg, respectively. The mean lymph node weight of the positive control group was 8.6 mg and which was 1.69 times greater than that of vehicle control group.

Stimulation Index (SI): The mean SI of the positive control group was 2.1 which satisfy the validity criteria of the study. The Sis of the 1, 10 and 25% test substance groups were 1.0, 1.3 and 1.0, respectively.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Migrated information

Conclusions:

BK-659 was considered to be a non-sensitizer and was classified to "classification not possible" for the hazard class of the GHS.

Executive summary:

Skin sensitization potential of BK-659 was assessed by Local lymph node assay: BrdU-ELISA, in female CBA/J mice.

This study was conducted in compliance with OECD Guideline for the Testing of Chemicals, No. 442B, "Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA" adopted on July 22, 2010.

In the pre-screen test, 0.5%, 1%, 2.5%, 5%, 10% and 25% test substance suspensions were applied to mice (one animal per dosage). As a result, no systemic toxicity or excessive irritation was observed in any treatment animals. Therefore, the dosages of the main study were set at 1%, 10% and 25%.

In the main study, acetone/olive oil (4:1 v/v%, AOO) was used as a vehicle control and 25.0% α-hexylcinnamaldehyde (HCA) were used as a positive control. Four animals were used in each group of the main study.

As a result, no abnormalities or no abnormal changes in body weights were observed in any treatment animals. Therefore, all animals were used for assessment of the skin sensitization potential. The Sis of the BK-659 treatment groups were all less than 1.6. Therefore, BK-659 was assessed to be a non-sensitizer.

In conclusion, under the condition tested, BK-659 was considered to be a non-sensitizer.