Pyridinium, 1-[2-[[4-[[3-[2-[6-amino-1-hydroxy-3-sulfo-5-[2-[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-2-naphthalenyl]diazenyl]-4-sulfophenyl]amino]-4-oxobutyl]sulfonyl]ethyl]-3-carboxy-, inner salt, sodium salt (1:4)

Endpoint summary

Description of key information

Skin sensitization

The stimulation index of the low dose, medium dose, and high dose group were 1.03, 0.96 and 0.58, respectively, and all < 1.6. Therefore, CR SB33 did not cause skin sensitization effect on CBA/CaJNarl mice.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 5, 2019 to September 16, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Species:

mouse

Strain:

other: CBA/CaJNarl

Sex:

female

Details on test animals and environmental conditions:

- Source: National Laboratory Animal Center
- Weight at study initiation: 18.6-23.1g
- Animal feeding situation: 1 mouse per cage (for preliminary test), 4 mice per cage (for definitive test).
- Acclimation period: 7 days
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 15%
- Frequemcy of ventilation: 10~15 times/hour.
- Photoperiod: 12-hrs dark / 12-hrs light

Vehicle:

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

Concentration:

50, 100, 200 mg/mL

No. of animals per dose:

For Control group: four
For Test group: sixteen

Details on study design:

Preliminary test: This study was comprised of four groups, a vehicle control (acetone : olive oil = 4 : 1, v/v) and three doe groups. Each group had one mouse. At the beginning of the study, the body weight of each mouse was measured and recorded. The control and test article were applied to the dorsum of two ears in each mouse with 25 µL. The clinical observation of the mice was conducted each day for 6 days, and the erythema and thickness for each mouse ears were recorded at day 1, day 3 and day 6.

Definitive test: This study was comprised of 5 groups, a vehicle control (acetone : olive oil = 4 : 1, v/v), a positive control (25% hexyl cinnamic aldehyde and 25% eugenol in acetone : olive oil = 4 : 1, v/v), a low dose group ( 50 mg/mL), a medium dose group (100 mg/mL) and a high dose group (200mg//mL). There were four mice in each group. The controls and test articles were applied to the dorsum of two ears in each mouse at day 1, day 2 and day 3 with 25 µL. The 25 mg/mL BrdU solution was administrated 0.2 mL through intraperitoneal injection at day 5. 24 hours after BrdU injection, sacrificed the mice by CO2.Excised the auricular lymph nodes from each mouse ear and process separately in PBS. The erythema and thickness were recorded before sacrificed. Preparation of cell suspensions: The lymph node from each mouse were excised by mechanical disaggregation through 200 micron-mesh sterile stainless steel gauze. The target volume was adjusted to 15 mL. The BrdU in proliferative cells was determined by the BrdU ELISA kit.

Positive control substance(s):

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

eugenol (CAS No 97-53-0)

Statistics:

1. Clinical observations were conducted during the study period and animal deaths should be documented.
2. Calculation of BrdU labeling index = (Absorbance of emission wave length OD 370 nm - Average of balnk absorbance of emission wavelength) - (Absorbance of reference wavelength OD 492 nm – Average of blank absorbance of reference wavelength).
3. Calculation of stimulation index (SI) = BrdU labeling index within vehicle control.
4. The result as positive when SI>=1.6. However, the dose-response relationship and statistical analysis including Duncan’s multiple range test of one-way ANNIVA and Dunnett’s test were used when SI between 1.6 and 1.9. A significant difference was defined as p<0.05.

Positive control results:

The stimulation index of the positive control group was 7.22. The stimulation index of the low dose, medium dose, and high dose groups were 1.03, 0.96 and 0.58, respectively.

Parameter:

SI

Remarks:

stimulation index

Value:

1

Test group / Remarks:

Vehicle control

Parameter:

SI

Remarks:

stimulation index

Value:

7.22

Test group / Remarks:

Positive control

Parameter:

SI

Remarks:

stimulation index

Value:

1.03

Test group / Remarks:

low dose

Parameter:

SI

Remarks:

stimulation index

Value:

0.96

Test group / Remarks:

medium dose

Parameter:

SI

Remarks:

stimulation index

Value:

0.58

Test group / Remarks:

high dose

Table 1. The body weight changes of the mice during study

Group	Animal ID	Body weight (g)		Weight changes^a(g)
		Day 1	Day 6
Vehicle control	01F	19.9	20.0	+0.1
	02F	21.5	21.5	+0.0
	03F	21.2	21.3	+0.1
	04F	21.2	21.2	+0.0
	Mean ±  SD	21.98± 0.70	22.20± 0.71
Positive control	05F	22.7	22.8	+0.1
	06F	22.8	22.8	+0.0
	07F	19.7	19.8	+0.1
	08F	20.7	20.8	+0.1
	Mean ± SD	22.38± 0.59	22.53± 0.62
Low dose	09F	21.1	21.2	+0.1
	10F	22.5	22.5	+0.0
	11F	23.1	23.2	+0.1
	12F	21.6	21.7	+0.1
	Mean ± SD	22.38 ± 0.69	22.55± 0.68
Medium dose	13F	21.0	21.0	+0.0
	14F	18.6	18.7	+0.1
	15F	21.5	21.5	+0.0
	16F	21.3	21.4	+0.1
	Mean ± SD	22.40 ± 0.32	22.65 ± 0.24
High dose	17F	22.0	22.0	+0.0
	18F	20.5	20.6	+0.1
	19F	18.8	18.9	+0.1
	20F	19.4	19.5	+0.1
	Mean ± SD	21.8 ± 0.61	22.03 ± 0.62

^aWeight changes = body weight (day 6) - body weight (day 1).

Table 2. Results of mice clinical observations

Group	Animal ID	Clinical observation
		Day 1	Day 2	Day 3	Day 4	Day 5	Day 6
Vehicle control	01F	N^a	N	N	N	N	N
	02F	N	N	N	N	N	N
	03F	N	N	N	N	N	N
	04F	N	N	N	N	N	N
Positive control	05F	N	N	N	N	N	N
	06F	N	N	N	N	N	N
	07F	N	N	N	N	N	N
	08F	N	N	N	N	N	N
Low dose	09F	N	N	N	N	N	N
	10F	N	N	N	N	N	N
	11F	N	N	N	N	N	N
	12F	N	N	N	N	N	N
Medium dose	13F	N	N	N	N	N	N
	14F	N	N	N	N	N	N
	15F	N	N	N	N	N	N
	16F	N	N	N	N	N	N
High dose	17F	N	N	N	N	N	N
	18F	N	N	N	N	N	N
	19F	N	N	N	N	N	N
	20F	N	N	N	N	N	N

^aN: normal 

Table 3. Thickness and erythema of mice

Group	Animal ID	Thickness (mm)		Erythema score^a
		Left ear	Right ear	Left ear	Right ear
Vehicle Control	01F	0.16	0.17	0	0
	02F	0.18	0.19	0	0
	03F	0.17	0.19	0	0
	04F	0.16	0.16	0	0
	Mean ± SD	0.17  ± 0.01		0.00 ± 0.0
Positive control	05F	0.38	0.45	3	4
	06F	0.37	0.47	3	4
	07F	0.40	0.37	4	4
	08F	0.37	0.45	3	4
	Mean ± SD	0.41  ± 0.04*		3.63  ± 0.5
Low dose	09F	0.26	0.21	0	0
	10F	0.20	0.21	0	0
	11F	0.21	0.22	0	0
	12F	0.20	0.20	0	0
	Mean ± SD	0.21  ± 0.02*		0.00 ± 0.0
Medium dose	13F	0.19	0.20	0	0
	14F	0.20	0.20	0	0
	15F	0.19	0.22	0	0
	16F	0.21	0.22	0	0
	Mean ± SD	0.20 ± 0.01*		0.00 ± 0.0
High dose	17F	0.23	0.21	0	0
	18F	0.22	0.22	0	0
	19F	0.21	0.22	0	0
	20F	0.23	0.22	0	0
	Mean ± SD	0.22 ± 0.01*		0.00 ± 0.0

^adepended on Erythema Scores to evaluate skin erythema.

* Thickness of ear significant difference with Vehicle control (p<0.05). Analyzed by Dunnett's test of one-way ANOVA.

Table 4. The level of BrdU

Group	BrdU labeling index ^a	stimulation index ^b
Vehicle control	0.129 ± 0.077	1.00
Positive control	0.931 ± 0.349*	7.22
Low dose	0.133 ± 0.062	1.03
Medium dose	0.123 ± 0.010	0.96
High dose	0.075 ± 0.020	0.58

^a BrdL labeling index = (Absorbance of emission wave length OD 370 nm - Average of balnk absorbance of emission wavelength) - (Absorbance of reference wavelength OD 492 nm – Average of blank absorbance of reference wavelength).

^b stimulation index (SI) = BrdU labeling index within vehicle control, each test article group and positive control / the mean BrdU labeling index within vehicle control.

*BrdU significant difference with vehicle control (p<0.05). Analyzed by Dunnett's test of one-way ANOVA.

Interpretation of results:

GHS criteria not met

Conclusions:

The study was conducted according to OECD 442B: 2018. The results indicated that there was no skin erythema of the mice ears in all groups. The ear thickness of test groups were significant increased compared to vehicle control group. BrdU labeling index of low, medium and high dose group were no significantly different with vehicle control group and showed 1.03, 096 and 0.58 in stimulation index, respectively. Therefore, the test article " CR SB33" under the conditions designed for this study, did not cause skin sensitization effect on CBA/CaJNarl mice.

Executive summary:

This test using the procedures outlined in the SuperLab Study Plan for MZ6-181200024 which is based on the SOP for the OECD 442B: 2018 and SuperLab standard operating procedure SOPP-347. CBA/CaJNarl female mice were used in this study. The study included vehicle control group, positive control group, low dose group (50mg/mL), divided into 5 groups randomly. There were 4 mice in each group. Control article or test article were applied on the dorsum of two ears in each mouse of each group on Day 1~3. BrdU solution was administered by intraperitoneal injection on Day 5. Ear thickness and erythema were recorded on Day 6, and the posterior ear lymph nodes were collected and prepared in phosphate buffer solution to cellular suspension. The BrdU content of each group was detected by ELISA kit and calculated the stimulation index (SI) of each group. The results showed that the mice in each group had no erythema on the ear skin on Day 6. The thickness of the ear in test groups were all significant increased compared with the vehicle control group. The stimulation index calculated showed that low, medium and high dose groups were 1.03, 0.96 and 0.58. The result was negative. Therefore, CR SB33 under the conditions designed for this study, did not cause skin sensitization effect on mice.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Skin sensitization

CBA/CaJNarl female mice were used in this study. The study included vehicle control group, positive control group, low dose group (50mg/mL), divided into 5 groups randomly. There were 4 mice in each group. Control article or test article were applied on the dorsum of two ears in each mouse of each group on Day 1~3. BrdU solution was administered by intraperitoneal injection on Day 5. Ear thickness and erythema were recorded on Day 6, and the posterior ear lymph nodes were collected and prepared in phosphate buffer solution to cellular suspension. The BrdU content of each group was detected by ELISA kit and calculated the stimulation index (SI) of each group. The results showed that the mice in each group had no erythema on the ear skin on Day 6. The thickness of the ear in test groups were all significant increased compared with the vehicle control group. The stimulation index calculated showed that low, medium and high dose groups were 1.03, 0.96 and 0.58. The result was negative. Therefore, CR SB33 under the conditions designed for this study, did not cause skin sensitization effect on mice.