2,2'-[[3-acetamido-4-[(2-chloro-4-nitrophenyl)azo]phenyl]imino]diethyl diacetate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04-AUG-1997 to 15-SEP-1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

OECD Guidelines for Testing of Chemicals, Number 406 "Skin Sensitization", adopted by the Council on July 17, 1992 (reported Paris, April 29, 1993).

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

Directive 96/54/EEC, B.6. "Skin Sensitization", July 30, 1996.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Study available is over 12 years old

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[[3-acetamido-4-[(2-chloro-4-nitrophenyl)azo]phenyl]imino]diethyl diacetate

EC Number:

216-251-6

EC Name:

2,2'-[[3-acetamido-4-[(2-chloro-4-nitrophenyl)azo]phenyl]imino]diethyl diacetate

Cas Number:

1533-78-4

Molecular formula:

C22H24ClN5O7

IUPAC Name:

2,2'-[[3-acetamido-4-[(2-chloro-4-nitrophenyl)azo]phenyl]imino]diethyl diacetate

Test material form:

solid

Details on test material:

Disperse Red 167:1

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

Test system: Albino Dunkin Hartley Guinea Pig, HsdPoc: DH, SPF
Rationale: Recognized by the international guidelines as a recommended test system (e.g. OECD, EEC).
Source: Harlan Nederland B.V.
Number of animals for main study/pretest: 15 females I 3 females, nulliparous and non-pregnant
Age at delivery: 5-7 weeks
Age at beginning of pretest/acclimatization period: 6- 8 weeks
Body weight at pretest start: Pretest groups: 376 - 423 g
Body weight at beginning of acclimatization period: Control and test group; 385-477 g
Identification: By unique cage number and corresponding ear tags.
Randomization: Randomly selected at time of delivery.
Acclimatization: One week for the control and test group under test conditions after health examination. One week for the animals of the pretest. Only animals without any visible signs of illness were used.

HUSBANDRY
Room no.: E 22 / RCC
Conditions: Standard Laboratory Conditions
Air-conditioned with 10-15 air changes per hour and continuously monitored environment with a target range for room temperature of 22 ± 3 °C and for relative humidity between 40-70% (values above 70% during cleaning process possible). The animals were provided with a 12-hour light, 12-hour dark cycle. Music was played during the light period.
Accommodation: Individually in Makrolon type-3 cages with standardsoftwood bedding ("Lignocel", Schill AG).
Diet: Pelleted standard Nafag Ecosan 845 25W4, batch nos. 37197 and 58197 guinea pig breeding / maintenance diet ("Nafag", Nahr- und Futtermittel AG), ad libitum. Results of analyses for contaminants are archived at RCC.
Water: Community tap water from Itingen, ad libitum. Once weekly additional supply of ascorbic acid (approx. 1 g/1) via the drinking water was provided. Results of bacteriological, chemical and contaminant analyses are archived at RCC.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

water

Concentration / amount:

5%

Day(s)/duration:

one administration on Day 1

Adequacy of induction:

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

Induction 2

Route:

intradermal

Vehicle:

other: Freud's adjuvant/saline (1:1 [v/v])

Concentration / amount:

5%

Day(s)/duration:

one administration on Day 1

Adequacy of induction:

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

Induction 3

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

50%

Day(s)/duration:

Day 8 / 48 hours

Adequacy of induction:

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

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

10%

Day(s)/duration:

Day 22 / 24 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Control group - 5 animals
Test group - 10 animals
Intradermal pretest - 1 animal
Epidermal pretest - 2 animals

Details on study design:

PRETEST / PERFORMED DURING THE ACCLIMATIZATION PERIOD
The objective of this investigation was to identify a maximally tolerated concentration of the test article suitable for the induction phase of the main study. In addition, a suitable non-irritant concentration of the test article, by the topical route of administration, was identified for the challenge application. The concentrations tested were for the epidermal application the most qualified to assure an optimum technical application procedure and for the intradermal injection the selected concentrations were tested up to 5 % (The Guinea Pig Maximization Test, page 270. Magnusson B.; Kligman A.M., 1969).
The procedure employed for these investigations was as follows:

INTRADERMAL INJECTIONS:
Four intradermal injections (0.1 ml/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of one guinea pig. One week later intradermal injections (0.1 ml/site) were made into the clipped flank of the same guinea pig at concentrations of 5, 3 and 1 % of the test article in hi-distilled water.
The resulting dermal reactions were assessed 24 hours later. For intradermal induction application in the main study a 5 % test article concentration was selected.

EPIDERMAL APPLICATIONS:
Four intradermal injections (0.1 ml/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of two guinea pigs. One week later both flanks of each of the guinea pigs were clipped and shaved just prior to the application. Thereafter 4 patches of filter paper (2 x 2 cm) were saturated with the test article at A= 50 % (this concentration was found to be the most qualified to assure an optimum technical application procedure), B = 25 %, C = 15% and D = 10% in hi-distilled water and applied to the clipped and shaved flanks. The volume of test article applied was approximately 0.2 g for the test article concentration of 50 % and 0.2 ml for the remaining concentrations.
The patches were covered by a strip of aluminium foil and firmly secured by elastic plaster wrapped around the trunk and covered with impervious adhesive tape. This procedure ensured the intensive contact of the test article. The dressings were removed after an exposure period of 24 hours.
Approximately 21 hours after removal of the dressing the application site was depilated with an approved depilatory cream (VEET Cream, Reckitt & Colman AG) to clean the application site from staining produced by the test article, so that possible erythema reactions were clearly visible at that time.
The depilatory cream was placed on the patch sites and surrounding areas, and left on for 3-5 minutes. It was then thoroughly washed off with a stream of warm, running water. The animals were then dried with a disposable towel, and returned to their cages.
The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema on a numerical basis according to Draize.
The allocation of the different test dilutions to the sites (A, B, C, D) on the animals was alternated in order to minimize site-to-site variation in responsiveness.
The concentration selected for the induction period and challenge procedure was 50 % and 10 %, respectively.

MAIN STUDY
INDUCTION
INTRADERMAL INJECTIONS I PERFORMED ON TEST DAY 1
An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 ml/site) were made at the border of a 4 x 6 cm area in the clipped region as follows:
Test Group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) The test article, diluted to 5 % with hi-distilled water.
3) The test article diluted to 5 %by emulsion in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

Control Group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) Bi-distilled water.
3) 1:1 (w/w) mixture of hi-distilled water in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

EPIDERMAL APPLICATIONS/ PERFORMED ON TEST DAY 8
One week after the injections, the scapular area (approximately 6 x 8 cm) was again clipped and shaved free of hair. A 2 x 4 cm patch of filter paper was saturated with the test article (50 % in hi-distilled water) and placed over the injection sites of the test animals. The volume of test article applied was approximately 0.3 g. The patch was covered with aluminium foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The dressings were left in place for 48 hours. The epidermal application procedure described ensured intensive contact of the test article.
The guinea pigs of the control group were treated as described above with bi-distilled water only.
Reaction sites were assessed for erythema and oedema 24 and 48 hours after removal of the dressing, using the numerical grading system according to Draize.

CHALLENGE / PERFORMED ON TEST DAY 22
The test and control guinea pigs were challenged two weeks after the epidermal induction application. The test and control guinea pigs were treated in the same way.
Hair was clipped and shaved from a 5 x 5 cm area on the left and right flank of each guinea pig just prior to the application. Two patches (2 x 2 cm) of filter paper were saturated with the highest non-irritating concentration of 10% (left flank) and the vehicle only (bi-distilled water applied to the right flank) using the same method as for the epidermal application. The volume of test article applied was approximately 0.2 ml. The dressings were left in place for 24 hours.
Approximately 21 hours after removal of the dressing the test sites treated with the test article were depilated as described in the epidermal pretest.
Approximately 24 and 48 hours after the removal of the dressing the application sites were assessed for erythema and oedema using the numerical scoring system according to Draize.

OBSERVATIONS
In addition to the sensitizing reactions the following observations and data were recorded during the test and observation period:
Viability / Mortality: Daily from delivery of the animals to the termination of test.
Clinical signs (local / systemic): Daily from delivery of the animals to the termination of test.
Skin reactions: At the times specified during the induction and challenge periods.
Body weights: At pretest start, one week prior to day 1 (main study), day 1 and termination of the test.
Records were maintained of all additional and standard observations.

PATHOLOGY
NECROPSY
Necropsy was performed by experienced prosectors in one animal (no. 777) of the control group which was found dead on test day 7. One animal (no. 793) of the pretest group was found dead during the acclimatization period (day -5).
The surviving animals were sacrificed by intraperitoneal injection of NARCOREN (Rhone Merieux GmbH, D-88471 Laupheim) at a dose of at least 5.1 ml/kg body weight (equivalent to 810 mg sodium pentobarbitone/kg body weight) and discarded.

Challenge controls:

The challenge site was evaluated 24 and 48 hours after the removal of the patch. The readings were made under artificial fluorescent light (daylight spectmm).
Redness constitutes the minimum criterion of an allergic reaction. Strongly sensitized animals display a vivid redness, associated with indurated swelling. The reactions were scored on the basis of the Draize score described under "Readings and Scoring".

Positive control substance(s):

yes

Remarks:

ALPHA-HEXYLCINNAMALDEHYDE

Results and discussion

Positive control results:

According to the procedures used in this experiment (performed from 06-JAN-1997 to 13-FEB-1997) positive results were observed in the treated animals after the epidetmal challenge application.
In the study 70 % of the animals of the test group were observed with positive skin reactions after treatment with a non-irritant test article concentration of 25 % in polyethylene glycol (PEG 400). No skin reactions were observed in the control group.
A response of at least 30% positive animals is considered positive "R43": may cause sensitization by skin contact according to the "Commission Directive 96/54/EEC, July 30, 1996 adapting to technical progress for the 22nd time Council Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances".
Therefore, the test article ALPHA-HEXYLCINNAMALDEHYDE applied at a concentration of 25 % in polyethylene glycol (PEG 400) is considered to be a sensitizer when used under the described test conditions.
According to the rating of allergenicity by Magnusson and Kligman the test article is a strong sensitizer.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10%

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

No symptoms of systemic toxicity were observed in the animals.

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10%

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

No symptoms of systemic toxicity were observed in the animals.

Results 3

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

4

Clinical observations:

One animal (no. 777) of the control group was found dead on test day 7.

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

4

Clinical observations:

One animal (no. 777) of the control group was found dead on test day 7.

Any other information on results incl. tables

PRETEST

 

The following reactions were observed in the pretest:

 

INTRADERMAL INJECTION / performed during the acclimatization period

Vehicle: Bi-distilled water

Animal No.	Sex	Concentration %	REACTION READINGS AFTER 24 HOURS
			Erythema	Oedema	Diameter (mm)
791	F	5	1	1	5 x 6
		3	1	1	5 x 5
		1	1	1	5 x 4

 

According to Magnusson – Kligman and to the findings observed, the concentration selected for the main study was 5%.

 

EPIDERMAL PRETEST / performed during the acclimatization period

Vehicle: Bi-distilled water

Animal No.	Sex	Concentration %	REACTION READINGS AFTER REMOVAL OF BANDAGE
			After 24 hours		After 48 hours
			E	Oe	E	Oe
792	F	50 25 15 10	1 0 1 0	0 0 0 0	1 0 1 0	0 0 0 0
793	F	10 50 25 15	0 EXITUS 0 0	0   0 0	0   0 0	0   0 0

E = Erythema

Oe = Oedema

 

Approximately 21 hours after removal of the dressing the test sites were depilated.

 

According to Magnusson – Kligman and the findings observed, the concentration selected for the induction period and challenge procedure was 50% and 10%, respectively.

 

MAUN STUDY – INDUCTION

 

CONTROL GROUP

SKIN RESPONSE AFTER THE EPIDERMAL APPLICATION OF THE VEHICLE (BI-DISTILLED WATER) DURING INDUCTION PERIOD (SCAPULAR AREA)

Animal No.	Sex	Erythema / Oedema Readings after removal of bandage
		24 hours		48 hours
		E	Oe	E	Oe
776 777 778 779 780	Female Female Female Female Female	0 EXITUS 0 0 0	0   0 0 0	0   0 0 0	0   0 0 0

E = Erythema

Oe = Oedema

 

TEST GROUP

SKIN RESPONSE AFTER THE EPIDERMAL APPLICATION OF FAT 41’021/A (50% IN BI-DISTILLED WATER) DURING INDUCTION PERIOD (SCAPULAR AREA)

Animal No.	Sex	Erythema / Oedema Readings after removal of bandage
		24 hours		48 hours
		E*	Oe	E*	Oe
781 782 783 784 785 786 787 788 789 790	Female Female Female Female Female Female Female Female Female Female	- - - - - - - - - -	0 0 0 0 0 0 0 0 0 0	- - - - - - - - - -	0 0 0 0 0 0 0 0 0 0

E = Erythema

Oe = Oedema

*Due to a red discoloration produced by the test article a possible erythema reaction could not be determined.

 

CONTROL GROUP

SKIN RESPONSE AFTER THE CHALLENGE APPLICATION OF BI-DISTILLED WATER (RIGHT FLANK)

Animal No.	Sex	Erythema / Oedema Readings after removal of bandage
		24 hours		48 hours
		E	Oe	E	Oe
776 777 778 779 780	Female Female Female Female Female	0 EXITUS 0 0 0	0   0 0 0	0   0 0 0	0   0 0 0

E = Erythema

Oe = Oedema

 

CONTROL GROUP

SKIN RESPONSE AFTER THE CHALLENGE APPLICATION OF FAT 41’021/A, 10% IN BI-DISTILLED WATER (LEFT FLANK)

Animal No.	Sex	Erythema / Oedema Readings after removal of bandage
		24 hours		48 hours
		E	Oe	E	Oe
776 777 778 779 780	Female Female Female Female Female	0 EXITUS 0 0 0	0   0 0 0	0   0 0 0	0   0 0 0

E = Erythema

Oe = Oedema

 

Approximately 21 hours after removal of the dressing the test sites were depilated.

 

TEST GROUP

SKIN RESPONSE AFTER THE CHALLENGE APPLICATION OF BI-DISTILLED WATER (RIGHT FLANK)

Animal No.	Sex	Erythema / Oedema Readings after removal of bandage
		24 hours		48 hours
		E*	Oe	E*	Oe
781 782 783 784 785 786 787 788 789 790	Female Female Female Female Female Female Female Female Female Female	0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0 0 0 0 0

E = Erythema

Oe = Oedema

 

TEST GROUP

SKIN RESPONSE AFTER THE CHALLENGE APPLICATION OF FAT 41’021/A, 10% IN BI-DISTILLED WATER (LEFT FLANK)

Animal No.	Sex	Erythema / Oedema Readings after removal of bandage
		24 hours		48 hours
		E*	Oe	E*	Oe
781 782 783 784 785 786 787 788 789 790	Female Female Female Female Female Female Female Female Female Female	0 0 0 0 0 0 1 0 0 0	0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0 1 0 0 0	0 0 0 0 0 0 0 0 0 0

E = Erythema

Oe = Oedema

 

Approximately 21 hours after removal of the dressing the test sites were depilated.

 

MACROSCOPIC FINDINGS

FEMALE

GROUP 1 (CONTROL GROUP)

ANIMAL 777     (SPONTANEOUS DEATH, 24-AUG-97)

LUNGS

NOT COLLAPSED. FOCUS/FOCI, ISOLATED, D=5 MM, DARK RED. DISCOLORATION, DARK RED

 

FEMALE

GROUP 4 (EPIDERMAL PRETEST)

ANIMAL 793     (SPONTANEOUS DEATH, 13-AUG-97)

LUNGS

FOCUS/FOCI, SEVERAL, D=10 MM, DARK RED.

 

 

BODY WEIGHTS (GRAM) SUMMARY

 

FEMALES

PRETEST		GROUP 1 CONTROL GROUP	GROUP 2 TEST GROUP	GROUP 3 INTRADERMAL PRETEST	GROUP 4 EPIDERMAL PRETEST
DAY 1 WEEK 1	MEAN ST. DEV. MINIMUM MAXIMUM N	-- -- -- -- 0	-- -- -- -- 0	376 -- 376 376 1	421 2.3 420 423 2

 

FEMALES

ACCLIMATIZATION		GROUP 1 CONTROL GROUP	GROUP 2 TEST GROUP	GROUP 3 INTRADERMAL PRETEST	GROUP 4 EPIDERMAL PRETEST
DAY 1 WEEK 1	MEAN ST. DEV. MINIMUM MAXIMUM N	413 20.2 387 444 5	439 27.6 385 477 10	405 -- 405 405 1	462 12.1 454 471 2

 

FEMALES

TREATMENT		GROUP 1 CONTROL GROUP	GROUP 2 TEST GROUP	GROUP 3 INTRADERMAL PRETEST	GROUP 4 EPIDERMAL PRETEST
DAY 1 WEEK 1	MEAN ST. DEV. MINIMUM MAXIMUM N	467 20.7 451 502 5	492 33.1 433 534 10	459 -- 459 459 1	493 -- 493 493 1

 

FEMALES

TREATMENT		GROUP 1 CONTROL GROUP	GROUP 2 TEST GROUP	GROUP 3 INTRADERMAL PRETEST	GROUP 4 EPIDERMAL PRETEST
DAY 29 WEEK 5	MEAN ST. DEV. MINIMUM MAXIMUM N	536 27.4 531 589 4	577 39.9 535 639 10	-- -- -- -- 0	-- -- -- -- 0

 

POSITIVE CONTROL RESULTS

 

ERYTHEMATOUS REACTIONS AFTER THE CHALLENGE PROCEDURE

	After 24 hours	After 48 hours
	Positive / total % of positive total	Positive / total % positive of total
CONTROL GROUP
ALPHA-HEXYLCINNAMALDEHYDE (left flank)	0 / 5 0	0 / 5 0
Polyethylene glycol (PEG 400) only (right flank)	0 / 5 0	0 / 5 0
TEST GROUP
ALPHA-HEXYLCINNAMALDEHYDE (left flank)	7 / 10 70	7 / 10 70
Polyethylene glycol (PEG 400) only (right flank)	0 / 10 0	0 / 10 0

No toxic symptoms were evidence in the guinea pigs of the control or test group.

No deaths occurred.

 

MAIN STUDY – CHALLENGE

TEST GROUP

SKIN RESPONSE AFTER THE CHALLENGE PROCEDURE OF ALPHA-HEXYLCINNAMALDEHYDE, 25% IN POLYETHYLENE GLYCOL (PEG 400) (LEFT FLANK)

Animal No.	Sex	Erythema/Oedema Readings after removal of bandage
		24 hours		48 hours
		E	Oe	E	Oe
381 382 383 384 385 386 387 388 389 390	Male Male Male Male Male Male Male Male Male Male	1 2 0 1 1 1 2 2 0 0	0 0 0 0 0 0 0 0 0 0	1 2 0 1 1 1 2 2 0 0	0 0 0 0 0 0 0 0 0 0

E = Erythema

Oe = Oedema

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item applied at a concentration of 10% in bidistilled water is considered not to be a sensitizer when used under the described test conditions. The substance is not classifiable according to CLP criteria.

Executive summary:

In order to assess the cutaneous allergenic potential of Disperse Red 167:1, the Maximization-Test in accordance with OECD Guideline No. 406 and the Directive 96154/EEC, B.6 was carried out in 15 (10 test and 5 control) female Albino guinea pigs.

 

The intradermal induction of sensitization was carried out with a 5 % dilution of the test article in bi-distilled water and in an emulsion with Freund's Complete Adjuvant (FCA)/physiological saline. The epidermal induction of sensitization was conducted under occlusion with the test article at 50 % in bi-distilled water. Two weeks after the epidermal induction application the challenge was completed by epidermal application of the test article at 10 % in bi-distilled water under occlusive dressing. The animals of the control group were induced with hi-distilled water and FCA/physiological saline and challenged similarly to those of the test group. Cutaneous reactions, i.e. erythema and eschar, as well as oedema formation were evaluated at 24 and 48 hours after removal of the dressing.

 

ERYTHEMATOUS REACTIONS AFTER THE CHALLENGE PROCEDURE

	After 24 hours	After 48 hours
	Positive / total % positive of total	Positive / total % positive of total
CONTROL GROUP
FAT 41’021/A (left flank)	0 / 4* 0	0 / 4 0
Bi-distilled water only (right flank)	0 / 4 0	0 / 4 0
TEST GROUP
FAT 41’021/A (left flank)	1 / 10 10	1/ 10 10
Bi-distilled water only (right flank)	0 / 10 0	0 / 10 0

* One animal (no. 777) of the control group was found dead on test day 7.

 

CONCLUSION

In this study 10% (1 out of 10) of the animals of the test group were observed with positive skin reactions after treatment with a non-irritant test article concentration of 10 % in bi-distilled water. No skin reactions were observed in the control group.

 

A response of at least 30% positive animals is considered positive "R43": may cause sensitization by skin contact according to the "Commission Directive 96/54/EEC, July 30, 1996 adapting to technical progress for the 22nd time Council Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances".

 

Therefore, the test item applied at a concentration of 10% in hi-distilled water is considered not to be a sensitizer when used under the described test conditions.