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Description of key information

CR SB37 was classified as positive reaction indicated that induced proliferation of lymphocytes in the lymph nodes (OECD TG442B).

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 November 15, 2017 to March 06, 2018
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
- Housing: four animals per cage
- Acclimation period: 7 days
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 15%
- Photoperiod: 12-hrs dark / 12-hrs light
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)
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.
Parameter:
SI
Value:
1.51
Test group / Remarks:
Low dose group (62.5 mg/ml)
Parameter:
SI
Value:
1.68
Test group / Remarks:
Medium dose group (125 mg/mL)
Parameter:
SI
Value:
2.26
Test group / Remarks:
High dose group (250 mg/mL)

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

Na

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).

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

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.

Justification for classification or non-classification