4-[bis(phenylsubstituted)substituted]phthalic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation: July 12, 2018; Completion of study: September 4, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 22, 2010

GLP compliance:

yes (incl. QA statement)

Type of study:

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

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

Animals:
Twenty four female CBA/J mice (7 weeks old) were obtained from Charles River Laboratories, Japan. The CBA/J is a sub-strain of CBA/JN which is the recommended strain in the OECD TG 442B and the test facility has LLNA historical control data of CBA/J.

At receipt, all animals showed good health conditions so they were received, assigned animal receipt numbers and weighed. The animals were acclimatized for 6 days after the receipt. No abnormalities were observed in any animal, the quarantine was completed and three animals were housed individually for pre-screen test. The remaining 21 animals were further acclimatized, and 13 days after receipt, were weighed, allocated to five groups (four per group) by body weight-stratified randomization and further acclimatized. The remaining animal and another two animals (derived from other studies) were used for additional pre-screen test. On 22 days after the receipt, 20 animals were weighed and confirmed to be within the range of mean body weight ±20% at the initiation of the main study. At the initial application, animals were 8 weeks old for the pre-screen test, 8 and 9 weeks old for additional pre-screen and 10 weeks old for the main study.

The animals were housed ≤10 animals per cage before the group allocation, 1 animal per cage for the pre-screen test (including additional pre-screen test) and 4 animals per cage after the group allocation for the main study. Clinical conditions and extretions were observed once or more daily: note that the animals for additional pre-screen test were observed once on the first sensitzation day (before sensitization). The animals were identified by painting on the tail with red ink before group allocation and by painting with other colors after the group allocation. 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 barrier-system animal rooms 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 a 7:00 and off at 19:00).
The animals were housed in polycarbonate cages with wood chips before and after the group allocation.
Animals had free access to a pelleted diet and 3 to 5 ppm chlorinate water via water bottles.

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Remarks:

Test substance dissolved at concentration of 50 w/v% in DMSO by ultrasonication. Prepared solution had not changed in visual inspection for 16 hours.

Concentration:

Pre-screen test: 1.00, 2.50, 5.00, 10.0. 25.0, 50.0 w/v%
Main test: 0.250, 0.500, 1.00 w/v%

No. of animals per dose:

Pre-Screen: 1 per dose
Main test: 4 per dose

Details on study design:

PRE-SCREEN TESTS (INCLUDING ADDITIONAL PRE-SCREEN TEST)
Objective: To determine appropriate dose levels for skin sensitization potential evaluation in the main study.

Grouping: The highest concentration was set at maximum possible concentration of 50 w/v%. The lower concentrations were determined according to the serial concentrations described in OECD TG 442B.
Group 1: 10.0 w/v% concentration
Group 2: 25.0 w/v% concentration
Group 3: 50.0 w/v% concentration
Group 4: 1.00 w/v% concentration
Group 5: 2.50 w/v% concentration
Group 6: 5.00 w/v% concentration

Preparation of test substance formulation:
On the first sensitization day of the pre-screen test, 0.50014 g of test substance was dissolved in DMSO by ultrasonication and filled up to 1 mL to make a 50.0 w/v% solution. The lower concentrations were prepared by serial dilution method.
Also on the first sensitization day of the additional pre-screen test, 0.50046 g of test substance was dissolved in DMSO by ultrasonication and filled up to 1 mL to make a 5.0 w/v% solution. The lower concentrations were prepared by serial dilution method.
The test substance solutions were stored in a refrigerator and use for 3 days.

Application: 25 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). Erythema scoring was evaluated according to the follow system, although no erythema was detected.

Observation Score
Very slight erythema (barely perceptible): 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness to eschar 4
formation preventing grading

Body weight:
Body weights of all the animals were measured on Day 1 and Day 6 using an electric balance.

Ear thickness:
The thickness of each ear was measured on Day 1 (before application), Day 3 and Day 6 using a resin vernier caliper. Thickness of each ear was measured in duplicate on each day and the mean values calculated.

Treatment of dead or moribund animals: No moribund or dead animals were observed.

Evaluation of result:
Dosages which induced the following signs were excluded from the main study.
- excessive irritation (Erythema score ≥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 and additional pre-screen test, >25% ear thickness increase, which indicated excessive irritation, was observed in 2.50 w/v% and above. Therefore, the dose levels of the main study were set at 1.00, 0.50 and 0.25 w/v%.

Grouping:
The test substance, vehicle control (DMSO) and positive control (25.0 w/v% HCA) groups were set in the main study. Four animals were used in each group.

Preparation of test substance, positive control substance and BrdU solution:
Test substance: On each sensitization day 0.1 g of test substance was dissolved in DMSO by ultrasonication and filled up to 10 mL to make a 1.00 w/v% solution. The 0.50 and 0.25 w/v% were prepared by serial dilution method.
Positive control substance solution: On the first sensitization day, 0.25036 g of HCA was dissolved in DMSO and filled up to 1 mL to make a 25.0% w/v% HCS solution under a yellow light condition. The solution was sub-divided into 3 air-tight containers and the containers were shaded and preserved in a refrigerator.
BrdU solution: Two days before the administration, 0.50002 g of 5-bromo-2'deoxyuridine (BrdU) was dissolved in physiological saline by ultrasonication and filled up to 50 mL to make a 10 mg/mL solution. The BrdU solution was filtrated by a sterilizing filter and was stored in a sterile container in a refrigerator until administration. The preparation was conducted under a yellow light condition, and the sterilization procedures were conducted in a clean bench.

Sensitization:
The vehicle, test substance formulation 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 of BrdU solution was administered intraperitoneally to each animal using a syringe and a needle.

Clinical observation:
Clinical observations were performed in the same way as the pre-screen test.

Body weight:
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 weight measurement of auricular lymph nodes:
Approximately 24 hours after the BrdU administration, 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 values and the standard deviations of the lymph node weights were calculated for each group. The lymph nodes were stored individually in a biomedical freezer.

BrdU labelling index:
The auricular lymph nodes were defrosted at room temperature, homogenized and suspended in physiological saline. This suspension was filtered by cell strainer and dispensed into 3 wells per animal of a 96 well microplate. The BrdU uptake levels were measured by ELISA using a commercial kit. Absorbance at 370 nm with a reference wavelength of 492 nm was measured using multidetection microplate reader. The mean value of the 3 wells was defined as BrdU labelling index.

Treatment of dead or moribund animals:
No dead or moribund animals were observed.

Positive control substance(s):

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

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks:

(mean of 4 animals)

Value:

3.48

Test group / Remarks:

Test substance at 0.250 w/v%

Results 2

Parameter:

SI

Remarks:

(mean of 4 animals)

Value:

3.4

Test group / Remarks:

Test substance at 0.500 w/v%

Results 3

Parameter:

SI

Remarks:

(mean of 4 animals)

Value:

4.3

Test group / Remarks:

Test substance at 1.00 w/v%

Results 4

Parameter:

SI

Remarks:

(mean of 4 animals)

Value:

2.33

Test group / Remarks:

Positive control

Results 5

Parameter:

SI

Remarks:

(mean of 4 animals)

Value:

1

Test group / Remarks:

Vehicle control

Cellular proliferation data / Observations:

PRE-SCREEN TEST:
Clinical signs: Thickening of auricle was observed in 10.0 w/v% treated animal and above on Days 5 and 6. Also thickening of auricle with scab formation was observed in 2.50 and 5.0 w/v% treated animals on Day 6. No abnormalities were noted in 1.00 w/v% treated animal.

Body weights: No abnormalities observed in any animal.

Thickness of auricle: >25% ear thickness increase was observed in 2.50 w/v% treated animal and above on Day 6. No abnormal changes were observed in 1.00 w/v% treated animal.

MAIN TEST:
Clinical signs: No abnormalities were noted in any group.

Body weights: No abnormal changes were noted in any treatment group compared to that of vehicle control group.

Auricular lymph node weights: The mean lymph node weights for the vehicle control group was 6.08 mg and thsoe for 1.00, 0.500 and 0.250 w/v% test substance groups were 14.00, 11.33 and 11.05 mg. The mean lymph node weights for the positive control group was 10.93 mg.

BrdU labelling indices and stimulation indices:
The mean BrdU labelling indices for the control group was 0.1685, and those for the 1.00, 0.500 and 0.250 w/v% of the test substance groups were 0.7240, 0.5735 and 0.5890, respectively. The BrdU labelling index for the positive control group was 0.3935. The SIs for the 1.00, 0.500 and 0.250 w/v% test substance groups were 4.30, 3.40 and 3.48. The SI for the positive control group was 2.33.

Any other information on results incl. tables

BrdU labelling indices and stimulation indices in the main study:

Exp. Group			Animal No.	Brdu labelling indices		Stimulation indices
Group	Substance name	Concentration (w/v%)		Individual	Mean ± S.E.	Individual	Mean ± S.E.
Vehicle control	DMSI	N.A	1	0.174	0.1685 ± 0.0134	1.0	1.00 ± 0.07
			2	0.145		0.9
			3	0.204		1.2
			4	0.151		0.9
Positive control	HCA	25.0	5	0.419	0.3935 ± 0.0097	2.5	2.33 ± 0.07
			6	0.390		2.3
			7	0.393		2.3
			8	0.372		2.2
Test substance	CIM-43	0.250	9	0.662	0.5890 ± 0.0357	3.9	3.48 ± 0.22
			10	0.578		3.4
			11	0.495		2.9
			12	0.621		3.7
		0.500	13	0.707	0.5735 ± 0.0538	4.2	3.40 ± 0.33
			14	0.565		3.4
			15	0.444		2.6
			16	0.578		3.4
		1.00	17	0.609	0.7240 ± 0.0436	3.6	4.30 ± 0.27
			18	0.776		4.6
			19	0.805		4.8
			20	0.706		4.2

 

 

 

 

 

 

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The SIs for the 1.00, 0.500 and 0.250 w/v% test substance groups were 4.30, 3.40 and 3.48. All the SIs were greater than 2.0. Therefore, under the conditions tested, the test substance was considered to be a sensitizer.

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 sensitization potential of CIM-43 was assessed by calculating the stimulation index.

In the pre-screen test, 50.0, 25.0, 10.0, 5.00, 2.50 and 1.00 w/v% test substance solutions, prepared with dimethyl sulfoxide (DMSO), were applied to mice daily for three consecutive days (one animal per dose level), and clinical observations, body weight measurements and ear thickness measurements were conducted. As a result, >25% ear thickness increase, which indicated excessive irritation, was observed in animals treated with 2.50 w/v% and greater concentrations. No abnormal changes which suggest excessive irritation or systemic toxicity were detected in animal treated at 1.00 w/v%. Thus, dose levels at 1.00, 0.500 and 0.250 w/v% were set for the main study.

In the main study, in addition to three test substance groups, a vehicle control group (DMSO) and positive control group ( α-hexylcinnamaldehyde (HCA)) were set. The vehicle, HCA or test substance was applied to the dorsal surface 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 sensitization, BrdU was administered to all the animals. Approximately 24 hours later, the auricular lymph nodes were collected, the BrdU uptake levels were measured by ELISA, and the SIs were calculated.

The SIs for the 1.00, 0.500 and 0.250 w/v% test substance groups were 4.30, 3.40 and 3.48. All the SIs were greater than 2.0. Therefore, under the conditions tested, the test substance was considered to be a sensitizer.