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Description of key information

Reactive Red 245 is a skin sensitiser

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:
24 Sept 1990 to 31 Jan 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes
Remarks:
Swiss GLP
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Data from a reliable in vivo test conducted before the enforcement of Commission Regulation (EU) 640/2012 of 06 July 2012 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) are available.
Specific details on test material used for the study:
Name: FAT 40406/A
Description: Solid, dark red powder

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: 6947/14
- Expiration date of the lot/batch: July, 1995

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the original container; protected from light; at room temperature (approx. 20 °C)
- Stability under test conditions: Stable for at least 48 hours
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.
- Age at acclimatization: 8 weeks
- Weight at acclimatization: 376-407g
- Housing: Individually in Makrolon type-3 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet: Pelleted standard 342 Kliba, Batch 59/90 and 60/90 guinea pig breeding/ maintenance diet ("Kliba", Klingentalmuehle AG, CH-4303 Kaiseraugst, ad libitum.
- Water: Community tap water from Itingen ad libitum
- Acclimation period: One week under test conditions after veterinary examination.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
Route:
intradermal
Vehicle:
physiological saline
Concentration / amount:
5%
Day(s)/duration:
Day 1
Adequacy of induction:
highest technically applicable concentration used
Route:
epicutaneous, open
Vehicle:
other: vaselinum album
Concentration / amount:
25 %
Day(s)/duration:
Day 8
Adequacy of induction:
other: non-irritant substance skin pre-treated with Sodium Lauryl Sulfate.
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: vaselinum album
Concentration / amount:
25 %
Day(s)/duration:
Day 21
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
other: vaselinum album
Concentration / amount:
10 % and 25 %
Day(s)/duration:
Day 35
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Ten females were treated with the vehicle alone and twenty females were treated with the test article.
Details on study design:
RANGE FINDING TESTS:
- Intradermal injections: Intradermal injections (0.1 mL/site) were made into the clipped flank of two guinea-pigs at concentrations of 1, 3 and 5 % of the test article in physiological saline. The resulting dermal reactions were assessed 24 h later.
- Epidermal applications: Patches of filter paper ( 2 x 2 cm) were saturated with concentrations of 5, 10, 15 and 25 % of the test article in vaselinum album 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. The dressings were removed after an exposure period of 24 h and the reaction sites were assessed immediately for erythema and edema . Further examination of the sites were performed 24 and 48 h after removal of the dressings. Prior to the first reading, the test sites were depilated to clean them from staining produced by the test article.

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: 1) Freunds' complete adjuvant 50:50 with physiological saline. 2) The test article, diluted to 5 % with physiological saline. 3) The test article diluted to 5 % by emulsion in a 50:50 mixture of Freund's complete adjuvant and physiological saline. Control Group: 1) Freund's complete adjuvant 50:50 with physiological saline. 2) physiological saline. 3) Freund's complete adjuvant 50:50 with physiological saline.
Epidermal applications: On day 7 of the test and approximately 24 h prior to the epidermal application the scapular area (approximately 6 x 8 cm) was clipped and shaved free of hair and the test areas were pretreated with 10 % Sodium-lauryl-Sulfate (SLS) in petrolatum-oil, because no primary irritation concentration could be determined in the corresponding pretest. The SLS was massaged into the skin with a glass rod without bandaging. This SLS-concentration enhances sensitization by provoking a mild inflammatory reaction. On day 8 of test a 2 x 4 cm patch of filter paper was saturated with the test article (25% in vaselinum album) 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. Reaction sites were assessed for erythema and edema immediately, 24 and 48 h after removal of the dressing.

B. CHALLENGE
FIRST CHALLENGE
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 vaselinum album) of the test article and b) with the vaselinum album 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 h later. The sites were assessed for erythema and edema immediately, 24 and 48 h after removal of the dressing. The control animals were treated in the same way as described above. Prior to the first reading, the test sites were depilated to clean them from staining produced by the test article.

SECOND 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 and an additional test article concentration was used (a - vehicle and 10 % test article in vaselinum album ; b - 25 % test article in vaselinum album). The control animals were treated with vaselinum album alone on the left flank.
Challenge controls:
The control animals were treated in the same way as described above without the test substance.
Positive control substance(s):
yes
Remarks:
A control group (Formaldehyde-solution) is tested twice a year for sensitivity check of the guinea pig strain. The most recent test was run from April 23 to May 24, 1990 (RCC 270066).
Positive control results:
According to the procedures used in this experiment (run from April 23 to May 24, 1990), clear positive results were observed in the formaldehyde solution treated animals after the epidermal challenge application (70 % positive).
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25%
No. with + reactions:
9
Total no. in group:
19
Remarks on result:
other: After First Challenge
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
7
Total no. in group:
19
Remarks on result:
other: After first challenge
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
25%
No. with + reactions:
7
Total no. in group:
19
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
7
Total no. in group:
19
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
10%
No. with + reactions:
1
Total no. in group:
19
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
10%
No. with + reactions:
1
Total no. in group:
19
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
20%
No. with + reactions:
7
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
20%
No. with + reactions:
3
Total no. in group:
10

SENSITIZING EFFECTS:


Control group:


No positive reactions were evident after the first and second challenge application, neither when treated with the vaseline album alone nor when treated with a 25 % test article dilution.


Test group:


Nine out of 19 animals of the first challenge and 7 out of 19 animals of the second challenge showed positive erythema reactions after the 24-hour reading when treated with a 25 % test article dilution. Seven out of 19 animals of the first and second challenge showed positive erythema reactions after the 48-hour reading when treated with the 25 % test article dilution. One out of 19 animals of the second challenge treated with 10 % test article dilution showed positive erythema reactions after the 24- and 48-hour reading. No positive erythema reactions for the both challenges treated with vaselinum album alone.


MORTALITY / VIABILITY: No spontaneous death occurred during the study. Female no. 317 was killed in extremis on day 16 of test due to ethical reason.


 


CLINICAL SIGNS (LOCAL):


Control group: Application area around the injection sites 1 and 3 was found to show erythema and edema from day 2 to 8; necroses were observed from day 10 to 13; encrustations from day 12 to 20 and exfoliation from day 19 to 39 (termination of test). No local symptoms were observed in injection site 2. In addition epidermal induction application area showed fissures from day 12 to 14 and first challenge application area showed staining from day 23 to 32.


 


Test group:


Application area around the injection site 1 was found to show erythema and edema from day 2 to 8; necroses were observed from day 10 to 12; encrustations on days 12 and 13; exfoliation from day 13 to 20 and fissures from day 19 to 39. Around the injection sites 2 and 3 edema were observed from day 2 to 8 and staining from day 2 to 12. Around the injection site 3 necroses were observed from day 10 to 12, encrustations on days 12 and 13, exfoliation from day 13 to 20 and fissures from day 19 to 39 while the injection site 2 showed fissures from day 33 to 39. In addition epidermal induction application area showed staining from day 10 to 31 and fissures from day 12 to 14; staining was observed in first and second challenge respectively from day 23 to 32 and 37 to 39. On day 9 of test no observation could be performed because the animals were bandaged semi-occlusively.


 


CLINICAL SIGNS (SYSTEMIC): Female no. 317 of test group: On day 13 of test: emaciated, sedated with ruffled fur, on day 14 of test: no amelioration of the state of health, on day 15 of test: no amelioration of the state of health. Oral administration four times in the course of the day of 10 ml mixture of water and glucose (diluted to 5 % in water). After the fourth administration a food consumption was observed. On day 16 of test: Slight amelioration of the state of health. Three oral administrations in the course of the day with the same 10 ml mixture of water/glucose 5 %. After the third administration a dyspnoea was observed. Due to ethical reason the animal was killed in extremis.


 


MICROSCOPICAL FINDINGS: Female no. 317: a) Nasosinusitis: Citrobacter diversus, Coagulase-negative, Staphylococci, Alpha-hemolysis inducing Streptococci and Branhamella caviae. b) Only Lactobacilli.


 


MACROSCOPICAL FINDINGS: Female no. 317: Lungs: no findings noted.


 


BODY WEIGHTS: Female no. 301 (control group) and females no. 321 and 326 (test group) slightly lost weight during the acclimatization period. The body weight gain of the further animals was not affected adversely during the study.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
FAT 40406/A is considered as skin sensitizer.
Executive summary:

In a GLP-compliant sensitivity study using the Maximization-Test, performed according to OECD guideline 406, guinea pigs were treated with the test substance. Twenty animals were treated with the test substance and ten animals with only the vehicle (physiological saline intradermally and vasiline for the epicutaneous application). The concentrations of the test substance used in the main study were determined by the results of the preliminary study. The intradermal induction of sensitization in the test group was performed using 5% of the test substance in both the vehicle and adjuvant/vehicle mixture. One week later this was boosted by the topical application of the test substance at 25 % concentration in vaselinum album over the injection sites. Animals of the control group were treated in the same manner but only the selected vehicle was used. Two weeks after the second induction all animals were challenged by topical application of the test substance at 25 % concentration in vaselinum album. Two weeks after the first challenge a second challenge was performed using 10 and 25 % of the test substance in vaselinum album. Nine and seven out of 19 animals reacted positive after 24 nad 48 h after the first challenge with 25% of the test substance. Seven animals reacted positive after 24 and 48 h after the second challenge with 25 % concentration. One out of 19 animals reacted positive after the challenge with 10 % of the test substance. Therefore it can be concluded that FAT 40406/A is a skin sensitizer.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

In a GLP-compliant sensitivity study using the Maximization-Test, performed according to OECD guideline 406, guinea pigs were treated with the test substance. Twenty animals were treated with the test substance and ten animals with only the vehicle (physiological saline intradermally and vasiline for the epicutaneous application). The concentrations of the test substance used in the main study were determined by the results of the preliminary study. The intradermal induction of sensitization in the test group was performed using 5% of the test substance in both the vehicle and adjuvant/vehicle mixture. One week later this was boosted by the topical application of the test substance at 25 % concentration in vaselinum album over the injection sites. Animals of the control group were treated in the same manner but only the selected vehicle was used. Two weeks after the second induction all animals were challenged by topical application of the test substance at 25% concentration in vaselinum album. Two weeks after the first challenge a second challenge was performed using 10 and 25 % of the test substance in vaselinum album. Nine and seven out of 19 animals reacted positive after 24 and 48 h after the first challenge with 25 % of the test substance. Seven animals reacted positive after 24 and 48 h after the second challenge with 25 % concentration. One out of 19 animals reacted positive after the challenge with 10 % of the test substance. Therefore, it can be concluded that FAT 40406/A is a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the findings in the skin sensitisation study, the Reactive Red 245 should be classified as skin sensitiser 1B according to EU CLP.