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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:
13.10.-13.11.2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Based on preliminary information about the substance irritancy potential the test Skin Sensitisation in Guinea Pigs was performed.

Test material

Constituent 1
Chemical structure
Reference substance name:
Trisodium 7-[[4-chloro-6-[(3-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]methylamino]-4-hydroxy-3-[(2-sulphonatophenyl)azo]naphthalene-2-sulphonate
EC Number:
274-418-9
EC Name:
Trisodium 7-[[4-chloro-6-[(3-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]methylamino]-4-hydroxy-3-[(2-sulphonatophenyl)azo]naphthalene-2-sulphonate
Cas Number:
70210-21-8
Molecular formula:
C26H20ClN7O10S3.3Na
IUPAC Name:
trisodium 7-[[4-chloro-6-[(3-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]methylamino]-4-hydroxy-3-[(2-sulphonatophenyl)azo]naphthalene-2-sulphonate
impurity 1
Chemical structure
Reference substance name:
Sodium chloride
EC Number:
231-598-3
EC Name:
Sodium chloride
Cas Number:
7647-14-5
Molecular formula:
ClNa
IUPAC Name:
sodium chloride
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Reactive Orange 5- Physical state: solid, powder- Analytical purity: 90% (w/w)- Impurities (identity and concentrations): NaCl (CAS: 7647-14-5) 10% (w/w)- Composition of test material, percentage of components:- Lot/batch No.: 8001- Expiration date of the lot/batch: unlisted- Storage condition of test material: The test substance should be stored in dry room in dark in closed container at the room temperature.

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: BFA
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Luboš Sobota, 298 03 Městec Králové, monitored breeding farm, RČH CZ 21760039- Age at receiving: 6-7 weeks - Weight at study initiation:- Housing: Sterilized shavings of soft wood, Monitored conditions, microbiologically defined background, according to internal SOP No.40- Diet: Pelleted standard diet for guinea pigs ad libitum – Altromin 3023 for Giunea pig - Water: Drinking tap water ad libitum (supplemented by ascorbic acid)- Acclimation period: 5 days-Number: 32 animals: -Sex: 12 males + 20 femalesENVIRONMENTAL CONDITIONSMicroclimatic conditions: Room temperature 22 ± 3°C, permanently monitoredRelative humidity 30 – 70 %, permanently monitoredLight: 12 hour light/dark cycle: 6am-6pm/6pm-6am

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
other: Natrii chloridum 0.9%, FCA, Vaseline album
Concentration / amount:
0,1 ml
Day(s)/duration:
0-6
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: vaseline
Concentration / amount:
20% test substance in vaseline
Day(s)/duration:
20
No. of animals per dose:
32 animals:Pilot experiment: 2 animalsExposed group: 20 animalsControl group: 10 animals
Details on study design:
MAIN STUDYA. INDUCTION EXPOSUREInduction: Intradermal InjectionsDay 0 – treated groupThree pairs of intradermal injections of 0.1 mL volume were given in the shoulder region, which was cleared of hair so that one of each pair lies on each side of the midline.Injection 1: a 1:1 mixture (v/v) Freunds Complete Adjuvant (FCA)/ physiological salineInjection 2: the test substance 30% in physiological salineInjection 3: the test substance 30% in a 1:1 mixture (v/v) FCA/ physiological salineDay 0 – control groupThree pairs of intradermal injections of 0.1 mL volume were given in the same sites as in the treated animals.Injection 1: a 1:1 mixture (v/v) Freunds Complete Adjuvant (FCA)/ physiological saline Injection 2: physiological salineInjection 3: physiological saline in a 1:1 mixture (v/v) FCA/ physiological salineInduction: Topical Application Day 6 - treated group24 hours before application (day 5) the test area was cleared of hair. A filter paper (2 x 4 cm) with 30% test substance in vaseline was applied to the test area and held in contact by an occlusive dressing for 48 hours. Day 6 – control group24 hours before application (day 5) the test area was cleared of hair. A filter paper (2 x 4 cm) with vaseline only was applied in a similar manner to the test area and held in contact by an occlusive dressing for 48 hours.B. CHALLENGE EXPOSURE- No. of exposures: 1- Day(s) of challenge: Day 20- Exposure period: 24h- Test groups: test substance in vaseline, vaseline- Control group: test substance in vaseline, vaseline- Site: R flank/L flank- Concentrations: 20% substance in vaseline- Evaluation (hr after challenge): 48h and 72hOBSERVATIONS:Clinical signs of intoxication and health condition: dailyMortality/viability: dailyBody weight: 0 and 24th dayThe skin reaction: 24, 48, 72, 96 and 120 hours after intradermal injection48, 72, 96, 120 hours after induction – topical application48 and 72 hours after challenge
Positive control substance(s):
yes
Remarks:
α-hexylcinnamaldehyde

Results and discussion

Positive control results:
Reliability of the experimental technique is checked periodically in about six-month interval by the experiment with known sensitiser α-hexylcinnamaldehyde. The result of last experiment (September 2015): positive skin reaction – 5 animals (total number of animals in exposed group = 10), i.e. 50%. According to the guideline the minimal number of animals with positive skin reaction should be 30%. The result is satisfactory.The results of reference study are described in Study No. 123/12/6MK- Ref/09/2015: Alfa-hexylcinnamaldehyd - Senzibilizace kůže, VUOS-CETA Report No. 15-511, 2015.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
20% test substance in vaseline
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no pathological reaction of skin
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
20% test substance in vaseline
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no pathological reaction of skin
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
20% test substance in vaseline
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no pathological reaction of skin
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
20% test substance in vaseline
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no pathological reaction of skin
Remarks on result:
no indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The exposed animals showed no pathological skin reactions and no other negative clinical symptoms throughout the experiment.The body weight of animals increased through the study and it was not affected by the treatment. In the course of experiment the animals did not show any clinical symptoms of intoxication.The test substance, Reactive Orange 5, was not shown to be a contact allergen in guinea pigs.
Executive summary:

The test substance, Reactive Orange 5, was tested for the assessment of skin allergic effects using albino guinea pigs (strain BFA).

The test was performed according to the EU Method B.6, Skin sensitisation.

The method is analogous to the OECD Test Guideline No. 406, Skin sensitisation.

The Magnusson and Kligman maximization procedure was followed. The pilot experiment was implemented on 2 animals.

The main test was performed on 20 treated and 10 control animals.

The experiment proceeded in three phases: two induction phases - topical application and the challenge phase. Potential skin reactions were evaluated at the end of experiment.

The evaluation of skin reactions in the exposed group carried out at 48 and 72 hours after the start of the challenge phase of study did not show any changes in skin of animals in both flanks.

No allergic skin reactions were observed incontrol group 48 hours and at 72 hoursfrom the start of application of the challenge dose.

No other negative clinical symptoms through the experiment were recorded.

The test substance, Reactive Orange 5,was not shown to be a contact allergen in guinea pigs.