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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:
January 15th to March 1st, 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Method was not available

Test material

Constituent 1
Chemical structure
Reference substance name:
6-chloro-2-[4-[ethyl(2-hydroxyethyl)amino]phenyl]-1-methylbenz[cd]indolium acetate
EC Number:
296-673-5
EC Name:
6-chloro-2-[4-[ethyl(2-hydroxyethyl)amino]phenyl]-1-methylbenz[cd]indolium acetate
Cas Number:
92952-73-3
Molecular formula:
C22H22ClN2O.C2H3O2
IUPAC Name:
6-chloro-2-{4-[ethyl(2-hydroxyethyl)amino]phenyl}-1-methylbenzo[cd]indolium acetate
Test material form:
solid: particulate/powder
Details on test material:
Basic Blue 145

In vivo test system

Test animals

Species:
guinea pig
Strain:
Himalayan
Remarks:
Ibm: GOHI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd. Wolferstrasse 4, CH-4414 Füllinsdorf
- Age at study initiation: males 7 weeks females: 8 weeks
- Weight at study initiation: males: 401 - 479 g females: 353 - 449 g
- Housing: Individually in Makrolon type-3 cages with standard softwood bedding
- Diet (e.g. ad libitum): Diet Pelleted standard Kliba 342 guinea pig breeding/ maintenance diet ("Kliba", Klingentalmühle AG, CH-4303 Kaiseraugst), ad libitum.
- Water (e.g. ad libitum): Community tap water from Itingen, ad libitum. Once weekly additional supply of ascorbic acid via the drinking water.
- Acclimation period: One week under test conditions after veterinary examination.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 3°C
- Humidity (%): 40-70 %
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light/12 hours dark

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: ethanol
Concentration / amount:
Test group: Three pairs of intradermal injections (0.1 ml/site)
1) Freund's complete adjuvant 50:50 with bi-distilled water.
2) The test article, diluted to 5% with ethanol.
3) The test article at the concentration used in (2), emulsified in a 50:50 mixture of Freund's complete adjuvant and the vehicle used in (2).
Control Group: Three pairs of intradermal injections (0.1 ml/site)
1) Freund's complete adjuvant 50:50 with bi-distilled water.
2) Vehicle used in (2) for test group.
3) Freund's complete adjuvant 50:50 with bi-distilled water.
Day(s)/duration:
Day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, open
Vehicle:
other: ethanol
Concentration / amount:
25 % / a 2 x 4 cm patch of filter paper was saturated with the test article
Day(s)/duration:
Day 8 - for 48 hours
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, open
Vehicle:
other: ethanol
Concentration / amount:
25 % / a 2 x 2 cm patch of filter paper, saturated with the test article
Day(s)/duration:
Day 22 - for 24 hours
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
other: ethanol
Concentration / amount:
25% / saturated filter paper (2 x 2 cm)
Day(s)/duration:
Day 36 - for 24 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
5 males, 5 females for the control group and 10 males, 10 females for the test group
Details on study design:
RANGE FINDING TESTS:
Intradermal concentration: 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.
Epidermal concentrations: Patches of filter paper (2 x 2 cm) were saturated with concentrations of 25%, 15%, 10% and 5% of the test article in ethanol and applied to the clipped and shaved flanks of each of four guinea-pigs. 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 for erythema and edema on a numerical basis according to the scale described above. Further examination of the sites were performed 24 and 48 hours after removal of the dressings.
Prior to the readings the test sites were depilated.


MAIN STUDY
A. INDUCTION EXPOSURE
Intradermal injections:
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) Freund's complete adjuvant 50:50 with bi-distilled water.
2) The test article, diluted to 5% with ethanol.
3) The test article at the concentration used in (2), emulsified in a 50:50 mixture of Freund's complete adjuvant and the vehicle used in (2).
Control Group:
1) Freund's complete adjuvant 50:50 with bi-distilled water.
2) Vehicle used in (2) for test group.
3) Freund's complete adjuvant 50:50 with bi-distilled water.

Epidermal applications:
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 (25% in ethanol) and placed over the injection sites of the test animals. The patch was covered by 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 des¬cribed ensured intensive contact of the test article.
Approximately 24 hours prior to the epidermal application the test area was be pretreated with 10 % Sodium-Lauryl-Sulfat (SLS) in petrolatum-oil. The SLS was massaged into the skin with a glass rod without bandaging. This concentration of SLS enhances sensitization by provoking a mild inflammatory reaction.
The guinea-pigs of the control group were treated as described above with the omission of test article.
Reaction sites were assessed for erythema and edema immediately, 24 and 48 hours after removal of the dressing

B. CHALLENGE EXPOSURE
The test and control guinea-pigs were challenged two weeks after the epidermal induction application.
Hair was clipped and shaved from a 5 x 5 cm area on the left and right flank of each guinea-pig. Two patches (2 x 2 cm) of filter paper were saturated with a) non-irritant concentration (25% in ethanol) of the test article and b) with the vehicle only and applied to the (a) left flank and (b) right flank using the same method as for the epidermal application.
The dressings were removed approximately 24 hours later. The sites were assessed for erythema and edema immediately, 24 and 48 hours after removal of the dres-sing, using the numerical scoring system as described under preliminary study.
Prior to the first reading, the test sites were depilated.
The control animals were treated in the same way as described above.

Re-challenge
A second challenge was performed two weeks after the first challenge.
The treatment procedure for the animals of the test group was similar as described for the first challenge with the exception that the flanks of all the guinea-pigs were changed (a - vehicle; b - test article dilution).
The control animals were treated with the vehicle alone on the left flank.


OTHER:
In addition to the sensitizing reactions the following observations and data were recorded during the test and observation period:
Mortality/Viability: Once daily.
Body Weights: At acclimatization start, start of application and termination of test.
Symptoms (local/systemic): Daily.
Challenge controls:
yes
Positive control substance(s):
yes
Remarks:
HCHO

Results and discussion

Positive control results:
clear positive results were observed in the HCHO treated animals after the epidermal challenge application in 90% of the animals

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25%
No. with + reactions:
6
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
5
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
15%
No. with + reactions:
9
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
etanol
No. with + reactions:
0
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
ethanol
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
25%
No. with + reactions:
5
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
4
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Only the 24-hour assessment in the first challenge met the cut-off value for a positive skin sensitization reaction. At all other evaluation time-points the threshold for a skin sensitiser was not met. Hence, the test substance was not considered to be a skin sensitiser.
Executive summary:

To assess the allergenic potential of the test substance in albino guinea pigs, the Maximization-Test of Magnusson & Kligman according to OECD Test Guideline 406 was conducted. Ten animals (5 males, 5 females) were used as control group and 20 animals (10 males, 10 females) were used as test group.

Prior to the first reading of the reactions, the skin was depilated to clean the application site from staining produced by the test article, so that the reactions (erythema) were clearly visible at that time.

The highest non-irritating concentration used for both challenge applications was 25%.

After the first challenge 6/20 (30%) animals showed a positive reaction at the 24-hour assessment and 5/20 (25%) at the 48-hour assessment. As no clear assessment for sensitizing properties can be made from the results of the first challenge (at least 30% positive animals), a second challenge with the same concentration was conducted in the same animals 14 days after the first challenge. After the re-challenge 5/20 (25%) animals showed a positive reaction at the 24-hour assessment and 4/20 (20%) at the 48-hour assessment.

No clinical signs were evident in the guinea pigs of either the control or test group. No deaths occurred.

Only the 24-hour assessment in the first challenge met the cut-off value for a positive skin sensitization reaction. At all other evaluation time-points the threshold for a skin sensitiser was not met. Hence, the test substance was not considered to be a skin sensitiser.