2-nitro-4-(trifluoromethyl)benzonitrile

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 Feb - 15 Mar 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted 17 Jun 2021

GLP compliance:

yes

Remarks:

The test complied with the Principles of Good Laboratory Practices (GLP) of the Certification and Accreditation Administration of the People’s Republic of China (2013 revised edition).

Type of study:

Buehler test

Justification for non-LLNA method:

The guinea pig is the animal of choice according to the Guidelines of Chemical Testing (HJ/T 153-2004) and Chemical Test Methods ¨C Health Effects Volume (second edition) and this test is the method of choice in China.

Test material

Test material information

Constituent 1

Reference substance name:

2-nitro-4-(trifluoromethyl)benzonitrile

EC Number:

212-298-1

EC Name:

2-nitro-4-(trifluoromethyl)benzonitrile

Cas Number:

778-94-9

Molecular formula:

C8H3F3N2O2

IUPAC Name:

2-nitro-4-(trifluoromethyl)benzonitrile

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Remarks:

Standard grade

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Liaoning Changsheng Biotechnology Co., Ltd., Liaoning, China
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 4 - 5 weeks
- Weight at study initiation: 248.33 - 303.43 g
- Housing: 5 - 6 guinea pigs were reared in one plastic box (L 92 cm x W 62 cm x H 25 cm)
- Diet: guinea pig maintenance feed (Liaoning Changsheng Biotechnology Co., Ltd., Liaoning, China); ad libitum
- Water: every-day drinking water; ad libitum
- Acclimation period: 7 days
- Indication of any skin lesions: no skin damage noticed

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.05 - 23.00
- Humidity (%): 40.15 - 69.96
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 / 12

- IN-LIFE DATES: 03 Feb - 15 Mar 2019

Study design: in vivo (non-LLNA)

Induction

Route:

epicutaneous, occlusive

Vehicle:

other: ethanol 80%

Concentration / amount:

0.2 g test sample and 0.2 mL vehicle

Day(s)/duration:

days 0, 7 and 14 (6 h exposure)

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: acetone

Concentration / amount:

0.2 mL 50 % test sample solution

Day(s)/duration:

day 28 (6 h exposure)

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

20 (in test group)
10 (controls)

Details on study design:

RANGE FINDING TESTS:
4 doses (unchanged test sample and 50%, 25% and 12.5% test sample solutions (W/V) in 80% ethanol) were prepared and tested in 2 animals. The exposure was 6 h (epicutaneous, occlusive), and skin response was observed approximately 1 and 24 h after removal of the dressing. Obvious erythema (H2) was noted in 1 animal exposed to the undiluted test sample. No visible change was observed for the other concentrations tested. Thus, 0.2 g (± 1%) test sample moistened with 0.2 mL vehicle was selected for the induction exposure, and 0.2 mL 50% test sample solution was selected for the challenge exposure.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 6 h
- Test groups: test substance moistened in 0.2 mL vehicle (80% ethanol)
- Control group: vehicle only
- Site: left side of the back
- Frequency of applications: every 7 days
- Duration: days 0 - 14
- Concentrations: undiluted test substance

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day of challenge: 28
- Exposure period: 6 h
- Test groups: test sample solution in acetone
- Control group: test sample solution in acetone
- Site: right side of the back
- Concentrations: 50%
- Evaluation (hr after challenge): approximately 24 h and 48 h after removal of the dressing

Challenge controls:

The control group is actually a challenge control.

Positive control substance(s):

yes

Remarks:

induction: 0.2 mL 50% 2-mercaptobenzothiazole solution (vehicle: 80% ethanol) challenge: 0.2 mL 2.5% 2-mercaptobenzothiazole solution (vehicle: acetone)

Results and discussion

Positive control results:

0.2 mL 50% 2-mercaptobenzothiazole solution (vehicle: 80% ethanol) was used for the induction exposure. 0.2 mL 2.5% 2-mercaptobenzothiazole solution (vehicle: acetone) was used for the challenge exposure. The skin sensitisation rate was 30%, confirming that the methodology used in this test and the sensitivity of the animals were valid.

In vivo (non-LLNA)

Results

Results 1

Reading:

other: reading validity test

Group:

positive control

Dose level:

Induction: 50% Challenge: 2.5%

Remarks on result:

other: positive indication of skin sensitisation (rate: 30%)

Results 2

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

Induction: 0% Challenge: 50%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

induction: undiluted (100%) challenge: 50%

No. with + reactions:

9

Total no. in group:

20

Clinical observations:

discrete erythema observed in 3 animals; confluent erythema observed in 6 animals

Remarks on result:

positive indication of skin sensitisation

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

Induction: 0% Challenge: 50%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Results 5

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

induction: undiluted (100%) challenge: 50%

No. with + reactions:

5

Total no. in group:

20

Clinical observations:

discrete erythema observed in 5 animals

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

SKIN RESPONSE

Induction: There were no visible changes in the skin at the exposure site for the animals in the control group. In the test group, all animals exhibited erythema, to varying degrees, at the skin of the exposure site. 9 animals exhibited hardening and breaking of the skin at the exposure site (see table 1).
Challenge: There were no visible changes at the skin of the exposure site in the animals in the control group. In the test group, 6 animals exhibited confluent erythema (grade 2) at the skin of the exposure site and 3 animals exhibited discrete erythema (grade 1) at the skin of the exposure site (see table 2; 24 h observation time point).

 

SKIN SENSITISATION RATE

Method for calculating skin sensitisation rate for all groups: The number of animals in this group exhibiting sensitivity response was divided by the total number of animals in this group, to arrive at the skin sensitisation rate for this group of animals.

Result: The skin sensitisation rate was 45%.

 

Table 1: Test Group Induction Exposure Skin Response

Animal no.	Day 0			Day 7			Day 14
	1 h after dressing was removed	24 h after dressing was removed	48 h after dressing was removed	1 h after dressing was removed	24 h after dressing was removed	48 h after dressing was removed	1 h after dressing was removed	24 h after dressing was removed	48 h after dressing was removed
1	H1	H1	N	N	N	N	H1	N	N
2	H2	H1	N	H2	H1	N	H2	H1	A
3	H1	N	N	H1	N	N	H1	N	A
4	H2	H1	N	H2	N	N	H2	H2	A
5	H2	H1	N	H1	H1	N	H1	N	N
6	H1	N	N	H1	N	N	H1	N	A
7	H2	H1	N	H1	N	N	H1	N	N
8	H2	H1	N	H2	N	N	H2	N	N
9	H1	N	N	H1	N	N	N	N	N
10	H2	H1	N	N	N	N	H2	N	N
11	H1	N	N	H1	N	N	H1	N	N
12	H2	H1	N	H2	N	N	H2	N	N
13	H1	N	N	H1	N	N	H1	N	A
14	H1	N	N	H2	N	N	H1	H1	N
15	H1	N	N	N	N	N	H1	N	N
16	H1	N	N	H1	N	N	N	N	A
17	H1	N	N	H1	H1	N	H1	N	N
18	H2	H1	N	H1	N	N	H1	H1	A
19	H1	N	N	N	N	N	H1	N	A
20	H1	N	N	H1	N	N	H2	H1	A

N: no visible changes H1: mild erythema H2: obvious erythema A: skin hardening and breaking

 

 

Table 2: Test Group Challenge Exposure (Day 28) Skin Response and Score

Animal no.	24 h after dressing was removed		48 h after dressing was removed
	Skin response	Total score	Skin response	Total score
1	N	0	N	0
2	N	0	N	0
3	B	2	A	1
4	B	2	N	0
5	A	1	N	0
6	N	0	N	0
7	B	2	A	1
8	N	0	N	0
9	N	0	N	0
10	N	0	N	0
11	N	0	N	0
12	N	0	N	0
13	B	2	A	1
14	B	2	A	1
15	A	1	N	0
16	N	0	N	0
17	A	1	N	0
18	N	0	N	0
19	N	0	N	0
20	B	2	A	1

N: no visible changes A: discrete erythema B: confluent erythema

 

BODY WEIGHT

All animals gained body weight during testing period.

Applicant's summary and conclusion

Interpretation of results:

other: Skin Sens. 1B (H317) according to Regulation (EC) No 1272/2008

Conclusions:

The skin sensitisation rate for the animals in the control group was 0%, and for the animals in the test group 45%. Under the conditions of this test and based on the test results, 2-nitro-4-(trifluoromethyl)benzonitrile is classified as Skin Sens. 1B (H317) according to Regulation (EC) No 1272/2008.