4,11-diamino-2-(3-methoxypropyl)-1H-naphth[2,3-f]isoindol-1,3,5,10(2H)-tetrone

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Data from a reliable in vivo test (non-LLNA method) conducted before the enforcement of Commission Commission Regulation (EU) 2017/706 of 19 April 2017 amending Annex VII to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) as regards skin sensitisation are available.

Test material

Test material information

Constituent 1

Reference substance name:

4,11-diamino-2-(3-methoxypropyl)-1H-naphth[2,3-f]isoindol-1,3,5,10(2H)-tetrone

EC Number:

235-402-7

EC Name:

4,11-diamino-2-(3-methoxypropyl)-1H-naphth[2,3-f]isoindol-1,3,5,10(2H)-tetrone

Cas Number:

12217-80-0

Molecular formula:

C20H17N3O5

IUPAC Name:

4,11-diamino-2-(3-methoxypropyl)-1H-naphtho[2,3-f]isoindole-1,3,5,10(2H)-tetrone

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

None

Specific details on test material used for the study:

None

In vivo test system

Test animals

Species:

guinea pig

Strain:

Himalayan

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd. Wölferstrasse 4, CH-4414 Füllinsdorf/Switzerland
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: Control and Test Group : 296 - 388 g; Pretest: 299 - 335 g
- Housing: Individually in Makrol on type-3 cages (size: 22x37x15 cm) with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet (e.g. ad libitum): Pelleted standard Kliba 342, Batches 78/93 (from acclimatization start to September 17, 1993) and 79/93 (from September 18, 1993 to termination of test) guinea pig breeding/ maintenance diet ("Kliba", Klingentalmühle AG, CH-4303 Kaiseraugst), ad libitum.
- Water (e.g. ad libitum): Community tap water from Füllinsdorf, ad libitum. Once weekly additional supply of ascorbic acid (1 g/1) via the drinking water. Results of bacteriological, chemical and contaminant analyses are included in this report
- Acclimation period: One week for the control and test group under test conditions after health examination. No acclimatization period for the animals of the pretest. Only animals without any visual signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 ± 3
- Humidity (%):40-70
- Air changes (per hr): Air-conditioned with 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light (approx. 100 lux) /12 hours dark, music during the light period.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

other: ethanol

Concentration / amount:

5 %

Day(s)/duration:

day 1

Adequacy of induction:

highest technically applicable concentration used

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

other: ethanol

Concentration / amount:

25 %

Day(s)/duration:

day 8

Adequacy of induction:

non-irritant substance, but skin pre-treated with 10% SDS

Challenge

Challenge 1

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: ethanol

Concentration / amount:

25 %

Day(s)/duration:

day 22

Adequacy of challenge:

highest non-irritant concentration

Challenge 2

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

other: ethanol

Concentration / amount:

25 %

Day(s)/duration:

day 29

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Main study:
30 females
Pretest:
6 females

Details on study design:

RANGE FINDING TESTS:
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 procedure employed for these investigations was as follows:

INTRADERMAL INJECTIONS:
Intradermal injections (0.1 ml/site) were made into the clipped flank of two guinea-pigs at concentrations of 5, 3 and 1% of the test article in ethanol. The resulting dermal reactions were assessed 24 hours later. For intradermal induction application a 5 % test article dilution was selected.

EPIDERMAL APPLICATIONS:
Both flanks of each of 4 guinea pigs were clipped and shaved just prior to the application. Thereafter, patches of filter paper (2x 2 cm) were saturated with concentrations of 5, 10, 15 and 25 % of the test article in ethanol and applied to the clipped and shaved flanks. The patches were covered by a strip of aluminum 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 and the reaction sites were assessed 24 hours 20 minutes and 48 hours 20 minutes after removal of the bandage for erythema and oedema on a numerical basis according to Draize described above.
21 hours after removing of the dressing, the application site was depilated with an approved depilatory cream (VEET Cream, Reckitt & Col mann AG, CH-4005 Basel) to clean the application site from the dark-blue staining produced by the test article, so that possible erythema reactions were clearly visible at that time. The depilatory 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.

MAIN STUDY
A. INDUCTION EXPOSURE
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 ethanol.
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) Ethanol.
3)*1:1 (v/v) mixture of ethanol in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

Epidermal applications
On test day 7 and approximately 17 hours prior to the epidermal application the scapular area (approximately 6 x 8 cm) was clipped, shaved free of hair and the test area was pretreated with 10 % Sodium-Lauryl-Sulfate (SLS) in paraffinum perliquidum as no primary irritation had been observed in the pretest. The SLS was massaged into the skin with a glass-rod without bandaging. This 10% concentration of SLS enhances sensitization by provoking a mild inflammatory reaction. On test day 8, a 2 x 4 cm patch of filter paper was saturated with the test article (25 % in ethanol) and placed over the injection sites of the test animals. The patch was covered with aluminum 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 ethanol 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

B. CHALLENGE EXPOSURE
FIRST 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 (2x 2 cm) of filter paper were saturated with the highest non-irritant concentration of 25 % (left flank) of test article in ethanol and the vehicle only (ethanol, applied to right flank) using the same method as for the epidermal application. 21 hours 30 minutes after removing of the application the sites treated with the test article were depilated with an approved depilatory cream (VEET Cream, Reckitt & Col mann AG, CH-4005 Basel). The cream was placed on the patch sites for 3- 5 minutes and then washed off with a stream of warm running water. When the application sites were clean and any stains from the test article removed the animals were dried with a disposable paper towel and returned to their cages. 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 the Draize.

SECOND CHALLENGE I performed on test day 29
A second challenge was performed one week after the first challenge. The treatment procedure was used for the animals in the test group and was the same as that described for the first challenge except the applications were made to the opposite flanks of the guinea pigs. In addition the test article at 15% in ethanol was applied to the posterior right flank.
The control animals were treated with the vehicle alone on the left flank.

Challenge controls:

Yes

Positive control substance(s):

yes

Remarks:

2-MERCAPTOBENZOTHIAZOL

Results and discussion

Positive control results:

None

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Intradermal induction: 5 %, epidermal induction: 25 %, first epidermal challenge: 25 %

No. with + reactions:

3

Total no. in group:

20

Clinical observations:

No clinical symptoms observed

Remarks on result:

other: First challenge

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Intradermal induction: 5 %, epidermal induction: 25 %, first epidermal challenge: 25 %

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

No clinical symptoms observed

Remarks on result:

other: First challenge

Results 3

Key result

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

Intradermal induction: 5 %, epidermal induction: 25 %, first epidermal challenge: 25 %, rechallenge (epidermal): 15 %

No. with + reactions:

2

Total no. in group:

20

Clinical observations:

No clinical symptoms observed

Results 4

Key result

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

Intradermal induction: 5 %, epidermal induction: 25 %, first epidermal challenge: 25 %, rechallenge (epidermal): 15 %

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

No clinical symptoms observed

Any other information on results incl. tables

The following reactions were observed in the pretest

Intra-dermal injection:

Vehicle: Ethanol

Animal NO	Sex	Concentration %	Reactions reading after 24 hours
			Erythema	Oedema	Diameter [mm]
347	F	5	-	1	5 X 5
348	F	5	-	1	11 X 14
347	F	3	-	1	9 X 11
348	F	3	-	1	10 X 12
347	F	1	-	1	9 X 8
348	F	1	-	1	12 X 10

* As the test article stained the skin blue, it was not possible to determine whether erythema was present.

The concentration used for the intradermal induction was 5%.

Epidermal Application:

Vehicle: Ethanol

Animal No / males	Sex	Concentration (%)	Reaction after 24 hrs (+/- 2 hrs)		Reaction after 24 hrs (+/- 2 hrs)
			Erythema	Edema	Erythema	Edema
349	F	25	0	0	0	0
		15	0	0	0	0
		10	0	0	0	0
		5	0	0	0	0
350	F	25	0	0	0	0
		15	0	0	0	0
		10	0	0	0	0
		5	0	0	0	0
351	F	25	0	0	0	0
		15	0	0	0	0
		10	0	0	0	0
		5	0	0	0	0
352	F	25	0	0	0	0
		15	0	0	0	0
		10	0	0	0	0
		5	0	0	0	0

CONTROL GROUP

First Challenge:

One animal was observed with a very slight erythematous reaction at the 24- and 48-hour reading when treated with the test article at 25% in ethanol. No reactions were noted with ethanol alone.

Dark-blue discolouration was noted on test day 23 after removal of the dressing. On test days 24 (24-hour reading) and 25 (48-hour reading) the discolouration was still observed on the test sites in spite of the depilation; however this discolouration had no influence on the evaluation of reactions.

Second Challenge:

No erythematous or oedematous reactions were observed in the control group treated with ethanol alone.

TEST GROUP

First Challenge:

Three animals (nos. 327, 343 and 344) showed a very slight erythematous reaction at the 24-hour reading when treated with the test article at 25% in ethanol.Only one animal (no. 327) still showed the same reaction at the 48-hour reading. No reactions were observed with ethanol alone.

Dark-blue discolouration was noted on test day 23 after removal of the dressing. On test days 24 (24-hour reading) and 25 (48-hour reading) the discolouration was still observed on the test sites in spite of the depilation; however this discolouration had no influence on the evaluation of reactions.

Second Challenge:

Animal no. 336 and animal no. 343 (this one showed an erythematous reaction at the first challenge) were observed with a very slight erythematous reaction at the 24-hour reading when treated with the test article at 25% in ethanol. No positive reations were evident in the animals, either when treated with ethanol alone or when treated with the test article at 15% in ethanol.

Dark-blue discolouration was noted on test day 23 after removal of the dressing. On test days 31 (24-hour reading), 32 (48-hour reading) and 33 the discolouration was still observed in spite of the depilation; however this discolouration had no influence on the evaluation of reactions.

CLINICAL SIGNS, SYSTEMIC

No symptoms of systemic toxicity were observed in the animals.

BODY WEIGHTS

The body weight gain of the animals was not affected adversely during the study.

VIABILITY / MORTALITY / MACROSCOPIC FINDINGS

As there were no deaths during the course of the treatment period no necropsies were performed.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

FAT 36152/F is considered to be a nonsensitizer in animals.

Executive summary:

The skin sensitization potential of FAT 36152/F in albino guinea pigs was evaluated according to the Maximization-Test of B. Magnusson and A.M. Kligman (1969) as described in OECD Guideline 406 and EU Method B.6.

Ten females were used in a control group and 20 females were used in a test group. The response of at least 30 % positive animals is considered positive "R43" following the "Commission Directive 91/325/EEC, March 1,1991, adapting to technical progress for the twelfth 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".

Ethanol was used a vehicle. Based on the results of the pre-test, intradermal and epidermal inductions were done at 1 and 25 % test concentrations. The highest non-irritating test article concentration used for the challenge application was 25 %. In addition in the second challenge the test article at 15% was applied.

First challenge

Three animals (nos. 327, 343 and 344) showed a very slight erythematous reaction at the 24-hour reading when treated with the test article at 25% in ethanol. Only one animal (no. 327) still showed the same reaction at the 48-hour reading. No reactions were observed with ethanol alone.

Second challenge:

Animal no. 336 and animal no. 343 (this one showed an erythematous reaction at the first challenge) were observed with a very slight erythematous reaction at the 24-hour reading when treated with the test article at 25% in ethanol. No positive reations were evident in the animals, either when treated with ethanol alone or when treated with the test article at 15% in ethanol..

Therefore, from the results described, FAT 36152/F is considered to be a non-sensitizer.