2-benzyl-2-methyl-3-butenitrile

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 25th -July 22nd, 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Test according to the method of B. Magnusson and A.M. Kligman (OECD-guideline 406 [1981] and EEC-guidelines 84/449/EEC [1984]) The test was performed according to the "OECD Principles of Good Laboratory Practice" in Testing of Chemicals, OECD, (Paris 1982).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

Positive Control: DNCB; 20 Control animals; Scoring system according to DRAIZE)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

yes

Remarks:

Positive Control: DNCB; 20 Control animals

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Test material

Test material information

Constituent 1

Reference substance name:

CITROWANIL B, Batch 890316, colourless, clear, viscous liquid

IUPAC Name:

CITROWANIL B, Batch 890316, colourless, clear, viscous liquid

Constituent 2

Reference substance name:

2-benzyl-2-methyl-3-butennitril

IUPAC Name:

2-benzyl-2-methyl-3-butennitril

Details on test material:

Citrowanil B

In vivo test system

Test animals

Species:

guinea pig

Strain:

Pirbright-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

Test animals: white Guinea-pigs, strain: Pribright, substrain: Bor: DHPW (SPF); source: Firma Winkelmann, Versuchstierzucht, Gartenstraße 30, D-4791 Borchen 1; Weight at the beginning of the study: 265 - 374 g; The used strain is sensitive to known allergenic compounds, which is based on repeated studies with positive control substanzes (e.g. DNCB).
Husbandry: Caging: max. 5 animals in one cage; Cagetype: Macrolon plastic cages IV (20 cm high, 33 cm width, 55 cm length); Lighting: Fluorescent light, 120 lux; Lighting period: 12 hours daily, from 7.00 a.m. to 7.00 p.m.; Temperature: 18 +/- 2 °C; Relative humidity: 50 - 85 %.
Food and feeding: Ssniff-G (Alleindiät für Meerschweinchen) ad libitum; Producer: Ssniff Spezialitäten GmbH.
Bedding: LIGNOCEL 3/4 Fasern (sterilisation at 180 °C, water binding capacity: 276.5 % of bedding); Producer: J. Rettenmaier & Söhne GmbH + Co., D-7092 Ellwangen-Holzmühle; Production: from pure soft wood, dried, disdusted and sterilized.
Water: ad libitum; System: Macrolon drinking bottles, 300 ml, Fa.Becker & Co., D-4629 Castrup-Rauxel; Quality: Aqua fontana as for human consumtion.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

arachis oil

Induction 2

Concentration / amount:

Intradermal induction: 10 % in vehicle and 10 % in FCA dilution
Epicutane induction: 10 % in vehicle
Epicutane challenge: 1 % in vehicle

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

arachis oil

Challenge 2

Concentration / amount:

Intradermal induction: 10 % in vehicle and 10 % in FCA dilution
Epicutane induction: 10 % in vehicle
Epicutane challenge: 1 % in vehicle

No. of animals per dose:

20

Details on study design:

Range Finding:
Two animals per group were treated dermally in a preliminary study under occlusiv conditions with the following concentrations (each 0.5 or 0.1 ml per animal) of the test sample:
dermal: 100 % (undiluted), 10 %, 5 %, 2.5 % and 1 % in aleum arachidis
intradermal: 10 %, 5 %, 2.5 % and 1 % in aleum arachidis

Main Study:
After an acclimatisation time of 7 days at least, the test was initiated. The guinea-pigs were divided into 2 groups (1 test- and 1 control-group) of 20 animals each (10 male and 10 female). Prior to treatement the shoulder of each animal was clipped with a small animal clipper (about 8x5 cm).
On the day of the experiment all animals of both groups were treated intradermally into two skin areas situated bilateral to the spine. The first injection sites were located at the craniodorsal area. The following injections were performed thereunder.
1. injection: 0.1 ml of FCA dilution
2. injection: 0.1 ml of Test sample in vehicle or for the control undiluted vehicle alone.
3. injection: 0.1 ml of Test sample in FCA dilution of for the control 10 % vehicle in FCA dilution
The adjuvant injections were made deep into the dermis to minimize sloughing.

7 days later the same sites were treated dermally with 0.5 ml of the test sample or vehicle. After the application the treated areas were covered with a gauze pad and fixed to the animals trunks with Elanoplast (Closed Patch Test). 48 h p.a. the bandage was removed.

3 weeks after the first intradermal treatment a second dermal treatment was carried out on both test- and control group as described above using "Hill-Top" Chambers. 0.5 ml of 1 % test sample in vehicle were applied to the left clipped flank of all animals and 0.5 ml of undiluted vehicle were applied to the right clipped flank of all animals. 24 h p.a. the bandages were removed.

One day thereafter the first evaluation was conducted (= 24 h value). 48 h after patch removing the 2nd evaluation (= 48 h value) was done.

Challenge controls:

1 % of the test sample in vehicle was the highest concentration without signs of primary irritation after epicutaneous administration according to the range finding test. Therefore a separate challenge control is not necessary.

Positive control substance(s):

yes

Remarks:

DNCB

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

1 % in vehicle

No. with + reactions:

1

Total no. in group:

20

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 1 % in vehicle. No with. + reactions: 1.0. Total no. in groups: 20.0.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

1 % in vehicle

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 1 % in vehicle. No with. + reactions: 0.0. Total no. in groups: 20.0.

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

undiluted vehicle

No. with + reactions:

1

Total no. in group:

20

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: undiluted vehicle. No with. + reactions: 1.0. Total no. in groups: 20.0.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

undiluted vehicle

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: undiluted vehicle. No with. + reactions: 0.0. Total no. in groups: 20.0.

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

1 % in Oleum arachidis

No. with + reactions:

10

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 1 % in Oleum arachidis. No with. + reactions: 10.0. Total no. in groups: 10.0.

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

1 % in Oleum acharidis

No. with + reactions:

10

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 1 % in Oleum acharidis. No with. + reactions: 10.0. Total no. in groups: 10.0.

Any other information on results incl. tables

Results of the Range finding test:

 

dermal(test sample diluted with oleum arachidis):

Animal No.	sex	Concentration	24 h	48 h
1	male	100 %	1	2
2	female	100 %	2	2
1	male	10 %	1	1
2	female	10 %	1	1
1	male	5 %	0	0
2	female	5 %	1	0
1	male	2.5 %	0	0
2	female	2.5 %	1	0
1	male	1 %	0	0
2	female	1 %	0	0

 

intradermal(test sample diluted with oleum arachidis):

Animal No.	sex	10 % le. + ri.	5 % le. + ri.	2.5 % le. + ri.	1 % le. + ri.
1	male *	2	2	1	1
2	female *	2	2	1	1

* 48 h post application the diameter of erythema was 8 mm.

 

Results of the Main Study:

 

Induction - intradermal:

Test group:

24 h p.a. slight to distinct erythemas and slight swellings were observed on the test group areas (test substance 10 %). These signs decreased distinctly until the next days p.a., so that from the 7^thday p.a. (= day of dermal treatment) no more findings were recorded.

The areas treated with the test substance + FCA and those treated only with FCA showed more or less distinct erythema and swelling. From the 5 - 6^thday p.a. these injection areas of all animals were burst open. From the 9^thday p.a. a healing up of the areas was recorded, so that at termination of the study all injection areas were completely healed up.

Control group:

On the areas of the control group (vehicle undiluted), only treated with the vehicle partly very slight erythemas and swellings were found, which decreased distinctly the following days so that on day 7 p.a. no more findings were observed.

The areas treated with the vehicle + FCA and those treated only with FCA showed more or less distinct erythema and swelling. From the 5 - 6^thday p.a. these injection areas of all animals were burst open. From the 9^thday p.a. a healing up of the areas was recorded, so that at termination of the study all injection areas were completely healed up.

Summary of response after intradermal induction treatment:

Group	area	day 1 - 6	day 7	day 9	termination
Test group	T	Erytema scoring: 1 – 2 Ø 9 – 10 mm, slight swelling	20/20 plane, n. sp. f.	n. sp. f.	n. sp. f.
	T + FCA	Erytema scoring: 1 – 2 Ø 9 – 10 mm, swelling	13/20 eschar 20/20 area bursted	16/20 area bursted eschar	areas closed
	FCA	Erytema scoring: 1 – 2 Ø 9 – 10 mm, swelling	20/20 area bursted	17/20 area bursted eschar	areas closed
Control group	V	Erytema scoring: 0 – 1 Ø 0 – 6 mm, 16/20 plane	20/20 plane, n. sp. f.	n. sp. f.	n. sp. f.
	V + FCA	Erytema scoring: 1 – 2 Ø 8 – 10 mm, slight swelling	20/20 area bursted	14/20 area bursted eschar	areas closed
	FCA	Erytema scoring: 2 Ø 8 – 10 mm, slight swelling	20/20 area bursted	16/20 area bursted eschar	areas closed

T      = Test sample (10 %)                  *… / 20  = of treated animals (areas)

V      = Vehicle (undiluted)                  n. sp. f.   = no specific findings

FCA   = Freund’s Complet Adjuvant

 

Induction - dermal:

Due to the above mentioned findings after the intradermal treatment an evaluation of findings was not possible in the animals.

 

Challenge - dermal:

24 h after challenge slight erythema was observed in 1/20 animals of the test group and 1/20 animals in the control group. 24 h later this erythema had completely disappeared. Since this finding, which was both slight and transitory, was evident in only 1 of the 20 animals in the test group and in the control group, it is not attributed any biological significance.

The control areas of both groups showed no findings at both time points.

Individual values after dermal treatment (induction and challenge): 

Test group			Induction Test sample 10 % in Ol. arachidis	Challenge right flank:  0.5 ml Ol. acharidis left flank:    0.5 ml 1 % Test sample in Ol. arachidis
Animal	sex	BW (g)	day 1-6 p.a.	24 hours, flank		48 hours, flank		BW (g)
No.		start		ri.	le.	ri.	le.	end
1	m	312	*	0	0	0	0	494
2	m	321	*	0	0	0	0	502
3	m	323	*	0	0	0	0	464
4	m	315	*	0	0	0	0	482
5	m	318	*	0	0	0	0	496
6	m	308	*	0	0	0	0	447
7	m	310	*	0	0	0	0	487
8	m	319	*	0	1	0	0	529
9	m	312	*	0	0	0	0	499
10	m	300	*	0	0	0	0	463
11	f	303	*	0	0	0	0	433
12	f	286	*	0	0	0	0	432
13	f	287	*	0	0	0	0	433
14	f	293	*	0	0	0	0	427
15	f	293	*	0	0	0	0	468
16	f	328	*	0	0	0	0	468
17	f	301	*	0	0	0	0	437
18	f	265	*	0	0	0	0	379
19	f	296	*	0	0	0	0	402
20	f	283	*	0	0	0	0	401

 

 

Control group			Induction Ol. arachidis undiluted	Challenge right flank:  0.5 ml Ol. acharidis left flank:    0.5 ml 1 % Test sample in Ol. arachidis
Animal	sex	BW (g)	day 1-6 p.a.	24 hours, flank		48 hours, flank		BW (g)
No.		start		ri.	le.	ri.	le.	end
1	m	318	*	0	0	0	0	466
2	m	374	*	0	0	0	0	468
3	m	339	*	0	0	0	0	524
4	m	370	*	0	0	0	0	509
5	m	345	*	0	0	0	0	516
6	m	327	*	0	0	0	0	520
7	m	360	*	0	0	0	0	526
8	m	331	*	0	0	0	0	502
9	m	364	*	0	1	0	0	547
10	m	322	*	0	0	0	0	460
11	f	300	*	0	0	0	0	423
12	f	322	*	0	0	0	0	428
13	f	327	*	0	0	0	0	426
14	f	300	*	0	0	0	0	449
15	f	277	*	0	0	0	0	383
16	f	315	*	0	0	0	0	433
17	f	311	*	0	0	0	0	417
18	f	281	*	0	0	0	0	405
19	f	267	*	0	0	0	0	379
20	f	311	*	0	0	0	0	401

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Migrated information

Conclusions:

Citrowanil B does not have sensitising properties.

Executive summary:

The experimental design was performed according to the method of B. Magnusson and A.M. Kligman (OECD / EEC-guidelines).

To test the sensitising properties of the compound 20 test- and 20 control-animals (guinea-pigs) were treated with 10 % of the test compound during the induction phases (intradermal and dermal) and with 1 % for the challenge (dermal) using Freund's Complet Adjuvant (FCA) to intensify the immunological reaction.

Any signs of erythema and edema were recorded 24 h and 48 h after the challenge.

According to the method of B. Magnusson and A.M. Kligman (OECD / EEC-guidelines), the compound "CITROWANIL B" is considered not to cause any hypersensitivity in guinea-pigs.