2,7-Naphthalenedisulfonic acid, 4-amino-5-hydroxy-3-[2-[3-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-6-[2-[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-, sodium salt (1:5)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 15, 2017 to March 06, 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)

GLP compliance:

yes

Type of study:

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

Test material

Test material information

Constituent 1

Reference substance name:

2,7-Naphthalenedisulfonic acid, 4-amino-5-hydroxy-3-[2-[3-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-6-[2-[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-, sodium salt (1:5)

EC Number:

815-135-8

Cas Number:

1386899-40-6

Molecular formula:

C26H25N5O22S7.5Na

IUPAC Name:

2,7-Naphthalenedisulfonic acid, 4-amino-5-hydroxy-3-[2-[3-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-6-[2-[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-, sodium salt (1:5)

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaJNarl

Sex:

female

Details on test animals and environmental conditions:

- Source: National Laboratory Animal Center
- Housing: four animals per cage
- Acclimation period: 7 days
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 15%
- Photoperiod: 12-hrs dark / 12-hrs light

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

62.5, 125 and 250 mg/mL

No. of animals per dose:

Four

Positive control substance(s):

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

eugenol (CAS No 97-53-0)

Results and discussion

Positive control results:

The BrdU labeling index of positive control group was 0.081 ± 0.007 and significantly higher than vehicle control group (ρ < 0.05). The stimulation index (SI) of positive control group was 2.06.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.51

Test group / Remarks:

Low dose group (62.5 mg/ml)

Results 2

Parameter:

SI

Value:

1.68

Test group / Remarks:

Medium dose group (125 mg/mL)

Results 3

Parameter:

SI

Value:

2.26

Test group / Remarks:

High dose group (250 mg/mL)

Any other information on results incl. tables

Table 1. Body weight of testing mice

Group	Animal ID.	Body Weight (g)		Body Weight Change (g)
		Day 1	Day 6
Vehicle control group	01F	20.5	20.6	+0.1
	02F	20.3	20.9	+0.6
	03F	20.5	20.7	+0.2
	04F	20.5	20.8	+0.3
Positive control group	05F	22.0	22.1	+0.1
	06F	20.9	21.0	+0.1
	07F	20.0	20.1	+0.1
	08F	20.1	21.0	+0.9
Low dose group	09F	19.6	20.0	+0.4
	10F	21.4	22.0	+0.6
	11F	20.6	20.6	+0.0
	12F	21.9	22.4	+0.5
Medium dose group	13F	20.5	21.0	+0.5
	14F	20.6	20.8	+0.2
	15F	21.7	21.9	+0.2
	16F	20.8	21.0	+0.2
High dose group	17F	20.7	20.9	+0.2
	18F	22.2	22.2	+0.0
	19F	20.8	21.0	+0.2
	20F	22.7	22.9	+0.2

Table 2.Clinical observation of testing mice

Group	Animal ID.	Clinical observation
		Day 1	Day 2	Day 3	Day 4	Day 5	Day 6
Vehicle control group	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 group	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 group	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 group	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 group	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. Ear thickness and skin erythema examination

Group	Animal ID.	Ear thickness (mm)^a		Erythema score
		Left-ear	Right-ear	Left-ear	Right-ear
Vehicle control group	01F	0.25	0.25	0	0
	02F	0.25	0.25	0	0
	03F	0.25	0.25	0	0
	04F	0.25	0.25	0	0
Positive control group	05F	0.25	0.25	0	0
	06F	0.25	0.25	0	0
	07F	0.25	0.25	0	0
	08F	0.25	0.25	0	0
Low dose group	09F	0.25	0.25	0	0
	10F	0.25	0.25	0	0
	11F	0.25	0.25	0	0
	12F	0.25	0.25	0	0
Medium dose group	13F	0.25	0.25	0	0
	14F	0.25	0.25	0	0
	15F	0.25	0.25	0	0
	16F	0.25	0.25	0	0
High dose group	17F	0.25	0.25	0	0
	18F	0.25	0.25	0	0
	19F	0.25	0.25	0	0
	20F	0.25	0.25	0	0

^aEar thickness was examined on day 6.

 

Table 4. BrdU content analysis

Group	BrdU labeling index	Stimulation index (SI)
Vehicle control group	0.039 ± 0.010	1.00
Positive control group	0.081 ± 0.007*	2.06
Low dose group	0.059 ± 0.025	1.51
Medium dose group	0.066 ± 0.026	1.68
High dose group	0.089 ± 0.014*	2.26

* Significant different from vehicle control group (ρ< 0.05).

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

According to OECD 442B test method that the stimulation index of high dose group (250 mg/mL) was ≥1.6 and statistical difference with the vehicle control group, CR SB37 was classified as positive reaction. Therefore, CR SB37 was classified as category 1B based on GHS criteria.

Executive summary:

This test using the procedures outlined in the SuperLab Study Plan for M62-170900010001EN which is based on the SOP for the OECD 442B (SOPP-347) and OECD 442B (OECD, 2010). The study included vehicle control group, positive control group, low dose group (62.5 mg/mL), medium dose group (125 mg/mL) and high dose group (250 mg/mL). Control article or test article were applied on the dorsum of two ears in each mouse of the group on day 1~3. BrdU solution was administered by intraperitoneal injection on day 5. Ear erythema was 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. On day 6, the mice in each group had no erythema on the ear skin and the thickness of the ear in dose group had no significant difference compared with the vehicle control group. The stimulus index of low and medium dose group were 1.51 and 1.68 and the BrdU labeling index was no significant difference compared with vehicle control group. The stimulation index of high dose group was 2.26 and the BrdU labeling was significantly higher than vehicle control group. Therefore, CR SB37 was classified as positive reaction under the test conditions.