2-isopropyl-N,2,3-trimethylbutyramide

Endpoint summary

Description of key information

In the key study (1976) equivalent or similar to OECD TG 406, ten albino guinea-pigs were used for the assessment of the sensitising potential of WS-23 (0.1%). The guinea pig maximisation test described Magnusson B. and Kligman A.M. (1970). Allergic Contact Dermatitis in the Guinea Pig, published by C. C. Thomas; Springfield, Illinois, was employed in this study for the detection of the delayed contact hypersensitivity in the guinea pig.

The procedure consists of two parts, i.e. induction and challenge.

In the induction exposure, the test animals were initially exposed to the test substance by intradermal injection and epidermal application.

No dermal irritation was observed following intradermal injection of containing WS-23 (0.1%) 5%w/v in v/v 1% aqueous ethanol alone. Reactions produced by the topically applied WS-23 (0.1%) 75% w/v in v/v 1% aqueous ethanol were obscured by the intradermal reactions which were still present at this time.

After two weeks from the induction period, the 5 x 5 cm area on the left flank of each animal was clipped from hair. 0.1 ml WS-23 (0.1%) 50% w/v in 1% v/v aqueous ethanol was applied to 2 x 2 cm of Watman No. 3MM paper in similar fashion to topical induction application, for 24 hours. 

The challenge was evaluated 24, 48 and 72 hours after removal of the patch. 

The extent and degree of ski reaction (erythema, oedema) to the challenge exposure in the test animals was evaluated. The results showed, that no erythrema and oedema were present during the time course of 24, 48 and 72 hours. One animal had dryness and sloughing of the epidermis after 72 hours, however without signs of erythema and oedema.

In conclusion, the results showed, that WS-23 (0.1%) did not produce any evidence of delayed contact hypersensitivity.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August - September 1976

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

OECD 406

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

no

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Available study report of 1976 according to Magnusson B. and Kligman A.M. (1970). Allergic Contact Dermatitis in the Guinea Pig, published by C. C. Thomas; Springfield, Illinois

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: WS-23 (0.1%)

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Redfern Animal Breeders Limited, Brenchley, Kent
- Housing: suspended cage in a wire mesh floor
- Food provided ad libitum (Vitamnin C enriched guinea-pig pelleted diet). Cabbage and hay were given once weekly.
- Water provided ad libitum

Route:

intradermal

Vehicle:

other: ethanol

Concentration / amount:

1. 0.1 ml Freunds complete adjuvant 50:50 with water for injection
2. 0.1 ml WS-23 (0.1%) 5% w/v in 1% v/v aqueous ethanol
3. 0.1 ml of a mixture containing WS-23 (0.1%) 5% w/v in 1% v/v aqueous ethanol and 50:50 Freunds complete adjuvant

Day(s)/duration:

one week

Adequacy of induction:

highest technically applicable concentration used

Route:

epicutaneous, occlusive

Vehicle:

other: ethanol

Concentration / amount:

0.40 ml of WS-23 (0.1%) 75% w/v in 1% v/v aqueous ethanol

Day(s)/duration:

48 hours

Adequacy of induction:

not specified

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: ethanol

Concentration / amount:

WS23 (0.1%) 50%w/v in 1% v/v aqueous ethanol

Day(s)/duration:

24, 48 and 72 hours

Adequacy of challenge:

not specified

No. of animals per dose:

10

Details on study design:

Delayed contact hypersensitivity in the guinea pig:

Preliminary investigations:
The intradermal and topical irritancy of a range of dilutions of WS-23 (0.1%) were investigated. The maximum concertation suitable for intradermal injection was found to be 5% w/v in 1% v/v aqueous ethanol, and for topical application to be 75% w/v in 1% v/v aqueous ethanol. These concentrations were subsequently utilised in the main study.

MAIN STUDY
A. INDUCTION EXPOSURE

Intradermal injections
- Exposure period: one week
- Test groups: 10 animals
- Site: dorsal skin 4X6 cm from the scapular region
- Frequency of applications: one application
- Concentrations: 0.1 ml WS23 (0.1%) 5% w/v in 1% v/v aqueous ethanol

Topical occlusive applications
- Exposure period: 48 hours
- Test groups: 10 animals
- Site: dorsal skin 4X6 cm from the interscapular area
- Frequency of applications: once
- Concentrations: 0.40 ml of WS-23 (0.1%) 75% w/v in 1% v/v aqueous ethanol

B. CHALLENGE EXPOSURE
- Exposure period: up to72 hours
- Day(s) of challenge: 24, 48 and 72 hours
- Test groups: 10 animals
- Site: 5X5 cm area on the left flank of each guinea-pig
- Concentrations: 0.1 ml of WS23 (0.1%) 50% w/v in 1% v/v aqueous ethanol
- Evaluation (hr after challenge): 3

Challenge controls:

no

Positive control substance(s):

no

Key result

Reading:

other: 24, 48 and 72 hours

Hours after challenge:

3

Group:

test chemical

Dose level:

WS-23 (0.1%) 50 % and 75% w/v in 1% v/v aqueous ethanol w/v in 1% v/v aqueous ethanol

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Erythema and oedema

Remarks on result:

no indication of skin sensitisation

Reading:

other: 24, 48 and 72 hours

Hours after challenge:

3

Group:

negative control

Dose level:

1% v/v aqueous ethanol

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

not specified

Remarks on result:

other: not specified

Reading:

other: 24, 48 and 72 hours

Hours after challenge:

3

Group:

positive control

Dose level:

Freunds complete adjuvant 50:50 with water for injection

Total no. in group:

10

Clinical observations:

not specified

Remarks on result:

other: not specified

Table 1. Dermal reactions elicited by the challenge application with WS-23 (0.1%) 50% w/v in 1% v/v aqueous ethanol
Guinea-pig Number	E = erythema O = Oedema	Score				Results Positive (+) Negative (-)
		24 hours		48 hours	72 hours
1	E	0	0		0	-
	O	0	0		0	-
2	E	0	0		0	-
	O	0	0		0	-
3	E	0	0		0	-
	O	0	0		0	-
4	E	0	0		0	-
	O	0	0		0	-
5	E	0	0		0	-
	O	0	0		0	-
6	E	0	0		0	-
	O	0	0		0	-
7	E	0	0		0	-
	O	0	0		0	-
8	E	0	0		0^#	-
	O	0	0		0	-
9	E	0	0		0	-
	O	0	0		0	-
10	E	0	0		0	-
	O	0	0		0	-

# Dryness and sloughing of the epidermis

Table 2. Magnusson and Kligman grading scale for the evaluation of challenge patch test reactions
0	No visible change
1	Discrete or patchy erythema
2	Moderate and confluent erythema
3	Intense erythema and swelling

Interpretation of results:

other: Not sensitising

Conclusions:

In this screening test, equivalent or similar to the OECD TG 406, performed in ten albino guinea-pigs, WS-23 (0.1%) did not produce any evidence of delayed contact hypersensitivity.

Executive summary:

In this study equivalent or similar to OECD TG 406, ten albino guinea-pigs of Hartley / Dunkin strain were used for the assessment of the sensitising potential of WS-23 (0.1%).

The method: guinea pig maximisation test described Magnusson B. and Kligman A.M. (1970). Allergic Contact Dermatitis in the Guinea Pig, published by C. C. Thomas; Springfield, Illinois was employed.

In the preliminary study, the intradermal and topical irritancy of a range of dilutions of WS-3 (0.1%) were investigated. The maximum concertation suitable for intradermal injection was found to be 5% w/v in 1% v/v aqueous ethanol, and for topical application to be 75% w/v in 1% v/v aqueous ethanol. These concentrations were subsequently utilised in the main study.

The procedure was divided into two parts, i.e. induction and challenge.

In the induction exposure, the test animals were initially exposed to the test substance by intradermal injection and epidermal application.

For intradermal injection, three pairs of intradermal injections containing: Freunds complete adjuvant, WS-23 (0.1%) 5%w/v in 1% aqueous ethanol and a mixture containing WS-23 (0.1%) 5%w/v in v/v 1% aqueous ethanol 50:50 with Freunds complete adjuvant, were made in the area of dorsal skin 4 x 6 cm from the scapular region clipped free from hair.

For the epidermal application, the same dorsal skin area 4 x 6 cm from the scapular region was clipped free from hair. A volume of 0.40 mL of WS-23 (0.1%) 75% w/v in v/v 1% aqueous ethanol was spread over a 3 x 6 cm patch of Watman No. 3MM paper and covered with the plastic impermeable adhesive tape. This in turn was firmly secured with the elastic adhesive bandage wound round the torso of the animal for 48 hrs.

No dermal irritation was observed following intradermal injection of containing WS-23 (0.1%) 5%w/v in v/v 1% aqueous ethanol alone.

Reactions produced by the topically applied WS-23 (0.1%) 75% w/v in v/v 1% aqueous ethanol were obscured by the intradermal reactions which were still present at this time.

After two weeks from the induction period, the 5 x 5 cm area on the left flank of each animal was clipped from hair. 0.1 ml WS-23 (0.1%) 50% w/v in 1% v/v aqueous ethanol was applied to 2 x 2 cm of Watman No. 3MM paper in similar fashion to topical induction application, for 24 hours.  

The challenge was evaluated 24, 48 and 72 hours after removal of the patch.  

The extent and degree of skin reaction (erythema, oedema) to the challenge exposure in the test animals was evaluated. The results showed, that no erythrema and oedema were present during the time course of 24, 48 and 72 hours. One animal (no. 8) had dryness and sloughing of the epidermis after 72 hours, however without signs of erythema and oedema.

In conclusion, the results showed, that WS-23 (0.1%) did not produce any evidence of delayed contact hypersensitivity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

The available data on skin sensitisation of the test substance, WS-23 do not meet the criteria for classification according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.