2-[(2-hydroxyphenyl)methyl]phenol; 2-[(4-hydroxyphenyl)methyl]phenol; 4-[(4-hydroxyphenyl)methyl]phenol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Tetsing was conducted between 16th January 2018 and 13th February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

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

Test material

Test material information

Constituent 1

Reference substance name:

2-[(2-hydroxyphenyl)methyl]phenol; 2-[(4-hydroxyphenyl)methyl]phenol; 4-[(4-hydroxyphenyl)methyl]phenol

EC Number:

908-912-9

Cas Number:

1333-16-0

Molecular formula:

C39H36O6

IUPAC Name:

2-[(2-hydroxyphenyl)methyl]phenol; 2-[(4-hydroxyphenyl)methyl]phenol; 4-[(4-hydroxyphenyl)methyl]phenol

Test material form:

solid

Details on test material:

2.1.2 Lot No. KZ517047
2.1.3 CAS No. 1333-16-0
2.1.4 Appearance Light pink or light yellow solid
2.1.5 Purity 97.4%
2.1.6 Expiration date - June 30th 2018
2.1.7 Storage condition (rom temperature 17.7 - 20.2 deg C)

Specific details on test material used for the study:

Test Substance

Name Bisphenol-F(Reaction mass of 2,2’- methylenediphenol and 4,4’-methylenediphenol and o-[(4-hydroxyphenyl) methyl]phenol)
Lot No. KZ517047
CAS No. 1333-16-0
Appearance Light pink or light yellow solid
Purity 97.4%
Expiration date Jun. 30, 2018
Storage condition Room temperature (Measurement value: 18.7– 20.2°C, Permissible range: 15–25°C)
Handling instruction Wear a mask, protective clothing, gloves and goggles

Supplier : KUKDO Chemical Co., Ltd.
Address 345-35, Kasan-dong, Kumchon-gu, Seoul, 08588, Republic of Korea

Date of receive Dec. 1, 2017

Residual test substance Any remaining test substance is discarded.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: KOATECH Co., Ltd., Republic of Korea
- Females (if applicable) nulliparous and non-pregnant: Not specified
Female, 13 mice, 8 weeks old, 20.8–22.7 g (Dose range finding study)
Female, 28 mice, 8 weeks old, 20.0–21.9 g (Main study)

- Microbiological status of animals, when known:

- Age and weight at study initiation:
Female, 13 mice, 8 weeks old, 20.8–22.7 g (Dose range finding study)
Female, 28 mice, 8 weeks old, 20.0–21.9 g (Main study)

- Housing: Polysulfone cage, 200Wx320Dx140H (mm)
Cages and feeders were washed in a cage washer and sterilized by an autoclave. 2–5 animals were stored per cage (during the quarantine-acclimation period) / 2–3 animals/cage (during the study)

- Diet (e.g. ad libitum): Pelleted rodent chow. The feed was placed in feeders and provided ad libitum.
- Water (e.g. ad libitum): Public tap water in Cheongju-si was filtered and irradiated by ultraviolet light and provided ad libitum in a quarantine room and by an automatic watering system in an animal room.
- Acclimation period:
Upon receipt, all animals were subjected to the clinical examination. Body weights were recorded using an electronic balance (CP622, Sartorius, Germany).
General health examinations were conducted by a staff veterinarian on the last day of the quarantine-acclimation period. All animals were observed for general condition and clinical signs daily and body weights were recorded on Day 8 after receipt. All animals were quarantined for 3 days. Then, they were moved from the quarantine room to the animal room and acclimated for 5 days.

During the acclimation period, a temporary identification number was marked on the tail using a red indelible pen. Each cage was attached with an individual coded card for the quarantine-acclimation period.
Following group assignment, the animals were uniquely identified by a blue indelible marking on the tail, and a color-coded cage card was placed on each cage displaying the group and dose levels.

- Group assignment:
Dose range finding study:
Following the quarantine-acclimation period (group assignment), 12 healthy females were selected and randomly distributed into 6 groups (2 females per group). The mean body weights of groups were calculated to ensure limited differences in each group.

Main study:
Following the quarantine-acclimation period (group assignment), 25 healthy females were selected and randomly distributed into 5 groups (5 females per group). The mean body weights of groups were calculated to ensure limited differences in each group.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6–23.6°C (Dose range finding study) 19.7–24.2°C (Main study)
- Humidity (%): 47.9–59.1% (Dose range finding study) 39.2–58.9% (Main study); Permissible range: 30.0–70.0%
- Air changes (per hr): 10–15 clean, fresh, filtered air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle (light between 7am and 7pm), 150–300 Lux

- IN-LIFE DATES:
Dose range finding study - 24th - 26th January 2018
Main study: 7th - 9th February 2018.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0, 25, 50 and 100% w/v

No. of animals per dose:

Range finding study:

Group Dose (%) Dose volume (μL/ear) Animals (ID No.)
G1 Negative control 0 (v/v) 25 2 (2101–2102)
G2 Test substance 1 5 (w/v) 25 2 (2201–2202)
G3 Test substance 2 10 (w/v) 25 2 (2301–2302)
G4 Test substance 3 25 (w/v) 25 2 (2401–2402)
G5 Test substance 4 50 (w/v) 25 2 (2501–2502)
G6 Test substance 5 100 (w/v) 25 2 (2601–2602)


Main study:
Group Dose (%) Dose volume (μL/ear) Animals (ID No.)
G1 Negative control 0 (v/v) 25 5 (2101–2105)
G2 Test substance 1 25 (w/v) 25 5 (2201–2205)
G3 Test substance 2 50 (w/v) 25 5 (2301–2305)
G4 Test substance 3 100 (w/v) 25 5 (2401–2405)
G5 Positive substance 25 (v/v) 25 5 (2501–2505)

Details on study design:

Dose Range Finding Study: The dose range finding part of this study was conducted under Non-GLP conditions.

Dose levels and administration: Dose selection was based on the consecutive doses and dose levels were selected from a series of appropriate concentrations such as 100, 50, 25, 10 and 5%.

Study method: The dose range finding study was conducted using the same method as in the main study, but lymph node proliferation measurement was not performed.

Justification for selection of dose levels in the main study: Based on the result of the dose range finding study, the high dose did not show toxicity, thus the high dose level for the main study was selected at 100% with two additional lower dose levels of 50 and 25%. In addition, the positive (25% HCA) and negative control (Vehicle) groups were included in the main study.

Main Study:
Clinical signs: All animals were observed for mortality, general condition and clinical signs for 6 days.

Disposition of dead animals: During the observation period, necropsy was not performed because there were no dead animals.

Body weights: Body weights were recorded prior to dosing (Day 1) and on the day of necropsy (Day 6).

Skin Sensitization Evaluated:
Ear thickness: Ear thickness measurement was taken using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6 (the day of necropsy).

Necropsy: Approximately 24 hours (24 h) after BrdU injection, the animals were euthanized under CO2 gas. Using a punch for skin biopsy of 6 mm in diameter, centering.

around both ears and avoiding the flexed part of the inner ear, the inner tissue of the ear was removed about 1 mm from the outside of the ear and tissues of both sides were weighed together. The draining auricular lymph nodes from each mouse ear were excised and processed separately in phosphate buffered saline (PBS, Lot No.: 6MB120, Lonza, U.S.A.) for each animal.

Preparation of cell suspension:
For each mouse, a single-cell suspension of lymph node cells (LNC) excised bilaterally was prepared by #70 nylon mesh to generate a single cell suspension. In each case, the target volume of the LNC suspension was adjusted to the determined optimized volume. The optimized volume (25 mL) was based on the mean absorbance within 0.1–0.2 in the negative control group.

Determination of cellular proliferation:
BrdU was measured by ELISA (PowerWave XS, BioTek Instruments, Inc., U.S.A.) using a commercial kit (Lot No.: 24890800, Roche Diagnostic GmbH, Mannhein, Germany). Briefly, 100 µ L of the LNC suspension was added to the wells of a flat-bottom microplate in triplicate. After dispensing, centrifugation (300 g, 10 min) was allowed the cells to settle to the bottom and remove the PBS. After fixation and denaturation of the LNC suspension, anti-BrdU antibody was added to each well and allowed to react. Subsequently, anti-BrdU antibody was removed by washing and the substrate solution was added and allowed to produce chromogen. Absorbance at 370 nm (Emission wavelength, em) with a reference wavelength of 492 nm (Reference wavelength, ref) was measured.

Results of absorbance of each well (Absorption, ABS) were used to calculate the BrdU labeling index by substituting into the following equation.

BrdU labelling index = (ABSem – ABS blankem) – (ABSref – ABS blankref)

em = Emission wavelength, em
ref = Reference wavelength, ref

The mean BrdU labelling index of each group was substituted into the following equation of stimulation index (Stimulation index, SI) to calculate SI.

SI = Mean of BrdU labelling index in the test substance / Mean of BrdU labelling index in the negative

Positive control substance(s):

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

Statistics:

Statistical analysis was conducted using a statistical program (version 9.3, SAS Institute Inc., U.S.A.) for the data including body weight, erythema score, ear thickness, ear weight and stimulation index.

Bartlett’s test was employed on homogeneity of variance (significance level: 0.05) for body weights, ear thickness, ear weight and stimulation index data. One-way analysis of variance (ANOVA) was employed on homogeneous data. Dunnett’s t- test was applied for multiple comparisons (significance levels: 0.05 and 0.01, one- tailed) between the negative control group (G1) and each of the test substance groups (G2–G4) or positive substance group (G5). Since not significant, Kruskal- Wallis test was employed on heterogeneous data and Steel’s test was applied for multiple comparisons (significance levels: 0.05 and 0.01, one-tailed) between the negative control group (G1) and each of the test substance groups (G2–G4) or positive substance group (G5).

Kruskal-Wallis test for the erythema score was employed on heterogeneous data, and Steel’s test was applied for multiple comparisons (significance levels: 0.05 and 0.01, one-tailed) between the negative control group (G1) and each of the test substance groups (G2–G4) or positive substance group (G5).

Results and discussion

Positive control results:

In the positive control group at 25%, the mean stimulation index was 5.12. There were significant increases when compared to the negative control group (p<0.05).

The positive control SI value was more 1.6, Therefore, these results were acceptable.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.86

Variability:

standard deviation of 1.01

Test group / Remarks:

25%

Results 2

Parameter:

SI

Value:

2.67

Variability:

standard deviation of 0.99

Test group / Remarks:

50% test item

Results 3

Key result

Parameter:

SI

Value:

2.9

Variability:

standard deviation of 0.65

Test group / Remarks:

100% test item

Results 4

Parameter:

SI

Value:

5.12

Variability:

standard deviation of 1.60

Test group / Remarks:

Positive control

Any other information on results incl. tables

Clinical signs:
There were no abnormal clinical signs or deaths in any dosing group during the observation period.

BodyWeights:

In the negative control group, the mean body weights were 23.8 and 23.7 g on Day 1 and Day 6 after dosing,respectively.

In the test substance groups at 25, 50 and 100%, the mean body weights were 23.3, 23.5 and 23.6 g on Day 1, and 23.5, 23.4 and 23.4 g on Day 6, respectively. There were no significant differences when compared to the negative control group.

In the positive control group at 25%, the mean body weights on Day 1 and Day 6  were

23.5 and 24.1 g, respectively. There were no significant differences when compared to the negative control group.

The table below shows the body weight data:

 

		Body weights (g)
Group/ Dose (%)	Time after administration(days)
		1	6
G1	Mean	23.8	23.7
0	S.D.	1.6	1.3
	N	5	5
G2	Mean	23.3	23.5
25	S.D.	0.8	1.0
	N	5	5
G3	Mean	23.5	23.4
50	S.D.	0.8	0.6
	N	5	5
G4	Mean	23.6	23.4
100	S.D.	1.5	1.1
	N	5	5
G5	Mean	23.5	24.1
25	S.D.	0.9	1.3
	N	5	5
G1:N,N-dimethylformamide, G2-G4: Bisphenol-F(Reaction mass of 2,2’-methylenediphenol and 4,4’-methylenediphenol and o-[(4- hydroxyphenyl) methyl]phenol) G5: α-hexylcinnamaldehyde
S.D.: Standard deviation
N: Number of animals
No statistically significant differences was noted in the substance groups from the negative control group (G1) (p>0.05, ANOVA).

Erythema score:

In the negative control group, the mean erythema score was 0 from Day 1 to Day 6 after dosing.

In the test substance groups at 25, 50 and 100%, the mean erythema scores were 0, 0 and 0 from Day 1 to Day 6, respectively. There were no significant differences when compared to the negative controlgroup.

In the positive control group at 25%, the mean erythema score was in the range of 0–2 from Day 1 to Day 6. There were significant increases when compared to the negative control group (p<0.05, Day 3, p<0.01: Days 4–6).

The table below shows the Erythema score:

1 2 3 4 5 6 G1 Mean 0 0 0 0 0 0 0 S.D. 0 0 0 0 0 0   N 5 5 5 5 5 5 G2 Mean 0 0 0 0 0 0 25 S.D. 0 0 0 0 0 0   N 5 5 5 5 5 5 G3 Mean 0 0 0 0 0 0 50 S.D. 0 0 0 0 0 0   N 5 5 5 5 5 5 G4 Mean 0 0 0 0 0 0 100 S.D. 0 0 0 0 0 0   N 5 5 5 5 5 5 G5 Mean 0 0 1 1 2 2 25 S.D. 0 0 0 0 1 0   N 5 5 5 # 5 ## 5 ## 5 ##

Group/ Dose(%)

                                                 Time after administration(days)                                                

 

G1:N,N-dimethylformamide,

G2-G4: Bisphenol-F(Reaction mass of 2,2’-methylenediphenol and 4,4’-methylenediphenol and o-[(4- hydroxyphenyl) methyl]phenol)

G5: α-hexylcinnamaldehyde S.D.: Standard deviation

N: Number of animals

#p<0.05, Significant difference from the negative control group (G1) by Steel's t-test.

##p<0.01, Significant difference from the negative control group (G1) by Steel's t-test.

Ear Thickness:

In the negative control group, the mean ear thickness value was 0.19–0.21 mm from Day 1 to Day 6 afterdosing.

In the test substance groups at 25, 50 and 100%, the mean ear thickness values (Day 1 to Day 6) were in the ranges of 0.19–0.21, 0.19–0.21 and 0.19–0.22 mm, There were significant increases when compared to the negative control group (p<0.01: Days 3 (50 and 100%), Day 6(100%)).

In the positive control group at 25%, the mean ear thickness value was in the range 0.19–0.23 mm from Day 1 to Day 6. There were significant increases when compared to the negative control group (p<0.01: Days 3 and 6).

The table below shows the Ear thickness results

Table 4. Mean Ear Thickness

 

		Ear thickness (mm)
Group/ Dose (%)		Time after administration (days)
		1	3	6
G1	Mean	0.19	0.20	0.21
0	S.D.	0.00	0.00	0.00
	N	5	5	5
G2	Mean	0.19	0.20	0.21
25	S.D.	0.00	0.00	0.00
	N	5	5	5
G3	Mean	0.19	0.21	0.21
50	S.D.	0.00	0.00	0.00
	N	5	5	5
			**
G4	Mean	0.19	0.22	0.22
100	S.D.	0.00	0.00	0.00
	N	5	5	5
			**	**
G5	Mean	0.19	0.22	0.23
25	S.D.	0.00	0.00	0.00
	N	5	5	5
			**	**
G1:N,N-dimethylformamide, G2-G4: Bisphenol-F(Reaction mass of 2,2’-methylenediphenol and 4,4’-methylenediphenol and o-[(4- hydroxyphenyl) methyl]phenol) G5: α-hexylcinnamaldehyde
S.D.: Standard deviation
N: Number of animals
**p<0.01, Significant difference from the negative control group (G1) by Dunnett's t-test.

 Ear Weights:

In the negative control group, the mean ear weight was 13.2 mg.

 

In the test substance groups at 25, 50 and 100%, the mean ear weights were 13.6, 13.7 and 15.0 mg, respectively. There were significant increases when compared to the negative control group (p<0.01 (100%)).

 

In the positive control group at 25%, the mean ear weight was 15.7 mg. There were significant increases when compared to the negative control group (p<0.01).

The table below shows the Mean Ear Weights:

 

Group/ Dose (%)		Ear Weights (mg)
G1	Mean	13.2
0	S.D.	0.2
	N	5
G2	Mean	13.6
25	S.D.	0.3
	N	5
G3	Mean	13.7
50	S.D.	0.8
	N	5
G4	Mean	15.0
100	S.D.	0.6
	N	5
		**
G5	Mean	15.7
25	S.D.	0.4
	N	5
		**
G1:N,N-dimethylformamide, G2-G4: Bisphenol-F(Reaction mass of 2,2’-methylenediphenol and 4,4’-methylenediphenol and o-[(4- hydroxyphenyl) methyl]phenol) G5: α-hexylcinnamaldehyde
S.D.: Standard deviation
N: Number of animals
**p<0.01, Significant difference from the negative control group (G1) by Dunnett's t-test.

Stimulation Index

(Figure 3, Tables 6, 14)

The mean stimulation index of the negative control group was 1.00.

 

In the test substance groups at 25, 50 and 100%, the mean stimulation indices of 1.86

2.67 and 2.90 were observed, respectively. There were significant increases when compared to the negative control group (p<0.05 (50 and 100%)).

 

In the positive control group at 25%, the mean stimulation index was 5.12. There were significant increases when compared to the negative control group (p<0.05). The positive control SI value was more 1.6,Therefore, these results were acceptable.

The table below shows the mean stimulation Index results for each group:

Group/ Dose (%)		BrdU labelling index	Stimulation index
G1	Mean	0.17	1.00
0	S.D.	0.04	0.21
	N	5	5
G2	Mean	0.33	1.86
25	S.D.	0.18	1.01
	N	5	5
G3	Mean	0.47	2.67
50	S.D.	0.17	0.99
	N	5	5
			#
G4	Mean	0.51	2.90
100	S.D.	0.11	0.65
	N	5	5
			#
G5	Mean	0.90	5.12
25	S.D.	0.28	1.60
	N	5	5
			#
G1:N,N-dimethylformamide, G2-G4: Bisphenol-F(Reaction mass of 2,2’-methylenediphenol and 4,4’-methylenediphenol and o-[(4- hydroxyphenyl) methyl]phenol) G5: α-hexylcinnamaldehyde
S.D.: Standard deviation
N: Number of animals
#p<0.05, Significant difference from the negative control group (G1) by by Steel's t-test.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In conclusion, the test substance, Bisphenol-F (Reaction mass of 2,2’-methylenediphenol and 4,4’-methylenediphenol and o-[(4-hydroxyphenyl)methyl]phenol), was considered to be a sensitizer on CBA/J mice under the conditions of this study.

Executive summary:

The purpose of this study was to assess the skin sensitization potential of the test substance, Bisphenol-F (Reaction mass of 2,2’-methylenediphenol and 4,4’-methylenediphenol and o- [(4-hydroxyphenyl) methyl]phenol), after application to the dorsum of each ear of female CBA/J mice.

The dose range finding study was conducted at dose levels of 100, 50, 25, 10 and 5% to determine the high dose level for the main study. Based on the result of the dose range finding study, the high dose did not show toxicity, thus the high dose level for the main study was selected at 100% with two additional lower dose levels of 50 and 25%. In addition, the positive and negative control groups were included in the main study.

 

As a result of the main study, no abnormal clinical signs or deaths were observed in any test substance group during the observation period.

In the test substance group at a dose of 25, 50 and 100%, the ear thickness, ear weight and stimulation index (SI) were significantly different when compared to the negative control group. And the body weight and erythema score were not significantly different when compared to the negative control group. The stimulation index (SI), which was an index of skin sensitization, was calculated to be >1.6 in all groups.

In the positive control group at 25%, the erythema score, ear thickness, ear weight and stimulation Index (SI) were significantly increased when compared to the negative control group, and the stimulation index (SI) was calculated to be 5.12, which was over 1.6.

 

In conclusion, the test substance, Bisphenol-F (Reaction mass of 2,2’-methylenediphenol and 4,4’-methylenediphenol and o-[(4-hydroxyphenyl) methyl]phenol), was considered to be a sensitizer on CBA/J mice under the conditions of this study.