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Toxicological information

Skin sensitisation

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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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Principles of method if other than guideline:
Guidelines followed
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

Constituent 1
Chemical structure
Reference substance name:
4,11-diamino-2-[3-(2-methoxyethoxy)propyl]-1H-naphth[2,3-f]isoindole-1,3,5,10(2H)-tetrone
EC Number:
265-334-3
EC Name:
4,11-diamino-2-[3-(2-methoxyethoxy)propyl]-1H-naphth[2,3-f]isoindole-1,3,5,10(2H)-tetrone
Cas Number:
65059-45-2
Molecular formula:
C22H21N3O6
IUPAC Name:
4,11-diamino-2-[3-(2-methoxyethoxy)propyl]-1H,2H,3H,5H,10H-naphtho[2,3-f]isoindole-1,3,5,10-tetrone
Test material form:
other: solid
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. Wolferstrasse 4, CH-4414 Füllinsdorf Switzerland
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: Control and Test Group: 332 - 428 g; Pretest: 335 - 405 g
- Housing: Individually in Makrolon 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/L) via the drinking water.
- 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: Air-conditioned with 10-15 air changes per hour
- Photoperiod: 12 hours artificial fluorescent light (approx. 100 lux) /12 hours dark, music during the light period.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: ethanol
Concentration / amount:
1 %
Day(s)/duration:
day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
other: ethanol
Concentration / amount:
25 %
Day(s)/duration:
day 7
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: ethanol
Concentration / amount:
25 %
Day(s)/duration:
day 22
Adequacy of challenge:
highest non-irritant concentration
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 1% 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 (2 x 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 and 48 hours 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 & Colmann AG, CH-4005 Basel) 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 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. A 25 % test article dilution was selected for the epidermal induction and challenge procedure.

MAIN STUDY
A. INDUCTION EXPOSURE
Intradermal injections 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 mixture (v/v) of Freund's complete adjuvant and physiological saline.
2) The test article, diluted to 1% with ethanol.
3)The test article diluted to 1 % by emulsion in a 1:1 (v/v) mixture of Freund's complete adjuvant and physiological saline.

Control Group: 1) 1:1 mixture (v/v) of Freund's complete adjuvant and physiological saline.
2) Ethanol.
3)1:1 mixture of ethanol and Freund's complete adjuvant 50:50 with physiological saline

Epidermal applications:
On test day 7 and approximately 24 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 per liquidum 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 approximately 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 the omission of test article (ethanol only). Reaction sites were assessed for erythema and edema 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 (2 x 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. Approximately 21 hours after removing of the application sites were depilated with an approved depilatory cream (VEET Cream, Reckitt & Colmann 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 edema 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 was the same as that described for the first challenge except the applications were made to the opposite flanks of the guinea pigs. The control animals were treated with the vehicle alone on the left flank.
Challenge controls:
Yes
Positive control substance(s):
not specified

Results and discussion

Positive control results:
None

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Group:
positive control
Remarks on result:
not measured/tested
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Remarks on result:
not measured/tested
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
Intradermal: 1 %, epidermal, challenge 1 and 2: 25 %
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No clinical signs were observed
Remarks on result:
other: Challenge 1
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
Intradermal: 1 %, epidermal, challenge 1 and 2: 25 %
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No clinical signs were observed
Remarks on result:
other: Challenge 1
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
Intradermal: 1 %, epidermal, challenge 1 and 2: 25 %
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No clinical signs were observed
Remarks on result:
other: Challenge 2
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
Intradermal: 1 %, epidermal, challenge 1 and 2: 25 %
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No clinical signs were observed
Remarks on result:
other: Challenge 2

Any other information on results incl. tables

OTHER SKIN EFFECTS


- Dark-blue discolouration was noted from test day 23 to 29 in the first challenge of the control.


- In the test group dark-blue discolouration was noted from test day 23 to 30 and (30 to 36) in the first and second challenge, respectively.


 


BODY WEIGHTS:


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


 


VIABILITY I MORTALITY I 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/G is considered to be a non-sensitizer.
Executive summary:

The purpose of this skin sensitization study was to assess the allergenic potential of FAT 36152/G when administered on the skin of albino guinea pigs. The highest concentration causing mild irritating when injected intradermally was determined to be 1 % based on the findings of a pre-test. Further, the results of epidermal testing during pre-test, showed the highest non-irritating test article concentration was 25 %, which was then used for epidermal induction (pre treatment with 10 % SDS), challenge 1 and rechallenge. No allergenic reactions were noted after the challenge 1 and rechallenge. From the results described above, FAT 36152/G was concluded to have no allergenic potency and considered to be a non-sensitizer.