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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 25 JULY 2018 to 31 AUGUST 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Constituent 1
Chemical structure
Reference substance name:
1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile
EC Number:
274-126-1
EC Name:
1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile
Cas Number:
69808-32-8
Molecular formula:
C17H17ClN4O2
IUPAC Name:
1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile
Test material form:
solid: particulate/powder
Details on test material:
- State of aggregation: powder
- Particle size distribution: not determined
- Mass median aerodynamic diameter (MMAD): not determined
- Geometric standard deviation (GSD): not determined
- Shape of particles: not determined
- Surface area of particles: not determined
- Crystal structure: not determined
- Coating: none
- Surface properties: not determined
- Density: 1.3526 ± 0.0010 g/cm3 at 20.0 ± 0.2°C
- Moisture content: <0.5%
- Residual solvent: not detected
- Activation: none
- Stabilisation: none
- Other:

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Elevage Janvier Labs (F-53941 Le Genest Saint Isle)
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known:
- Age at study initiation: 8 weeks old
- Weight at study initiation: yes
- Housing: the animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes. The temperature and relative humidity were controlled to remain within target ranges of 19ºC to 25ºC and 30% to 70% respectively. The rate of air exchange was at least ten changes per hour and the lighting was controlled by a time switch to give twelve hours continous light (07.00 to 19.00) and twelve hours darkness. The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: acclimatisation period of at least five days under stabling and nutritional conditions identical to those of the test.
- Indication of any skin lesions: none

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19ºC to 25ºC
- Humidity (%): 30% to 70%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light
- IN-LIFE DATES: From: 31 January To: 21 November 2018

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
50%, 25% and 10%
No. of animals per dose:
4 females mouse
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: 50% in propylene glicol (PG)
- Irritation: any signs of toxicity or excessive local irritation noted during this period were recorded.
- Systemic toxicity: any signs of toxicity or excessive local irritation noted during this period were recorded.
- Ear thickness measurements: ear thickness was recorded on day 1, day 3 and on day 6.
- Erythema scores: none

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA:BrdU
- Criteria used to consider a positive response: The Stimulation Index (SI) calculated by individual approach will determine the EC1.6 if the value is higher than 1.6.

TREATMENT PREPARATION AND ADMINISTRATION:
Three groups of four animals were treated for three consecutive days (D1, D2, D3) with 50 microlitters (25 microlitter per ear) of the test item diluted at concentrations of 10%, 25% and 50% in propylene glycol (PG). A further group of four animals was treated with PG.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Linear interpolation of points on the dose-response curve, immediately above and below the 1.6-fold threshold.

Results and discussion

Positive control results:
A study was performed to assess the sensitivity of the strain of mouseuse at these laboratories to a known sensitiser. The method was ddesigned to meet the requirements of the following:
- O.E.C.D. Guideline for the Testing of Chemicals Nº 442B "Skin sensitization: Local Lymph Node Assay: BrdU-ELISA" of July 22, 2010
-Test method B.51 Skin Sensitisation (Local Lymph Node Assay) - Council regulation No 640/2012 of 30 May 2012 (E.U. journal L193)
Test Item: alpha-Hexylcinnamaldehyde[101-86-0], 95%
Laboratory Project number: nºLLNA-BrdU-2018-B
Study dates: 04 July to 11 July 2018
Methods:
Three groups, each of four animals were treated with 50 microlitters (25 microlitters per ear) of alpha-Hexylcinnamaldehyde, as a solution in acetones/olive oil (4:1, v/v) at concentrations of 5%, 10% and 25% (v/v). A further control group of four animals was treated with acetone/olive oil (4:1, v/v) alone.
Results:
5% and 10% negative result. 25% positive result (1.61 +/- 0.20).
EC1.6 value: 24.66%for 5%, 10% and 25%.
Conclusion:
In conlcusion, in view of these results, under these experimental conditions, the substance alpha-Hexylcinnamaldehyde in accordance with the Regulation (EC) No. 1272/2008 has to be classified in category 1 "Skin sensitisation". The signal word "Warning" and hazard statement H317 "May cause an allergic skin reaction" are required.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
ca. 0.77
Variability:
+/- 0.13
Test group / Remarks:
#4 (50%)
Key result
Parameter:
SI
Value:
ca. 0.78
Variability:
+/- 0.14
Test group / Remarks:
#3 (25%)
Key result
Parameter:
SI
Value:
ca. 0.68
Variability:
+/- 0.14
Test group / Remarks:
#2 (10%)
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION
All the SI calculations are below 1.6 so, the results are negative in all the cases. See "Any other information on results incl. tables".

CLINICAL OBSERVATIONS:
None


BODY WEIGHTS
All the groups gained weight. See "Any other information on results incl. tables".

Any other information on results incl. tables

DETAILS ON STIMULATION INDEX CALCULATION






























































































































































GroupsTest itemAnimal No.BrdU-index (DO indiv)BrdU-index (DO mean)BrdU-index mean*Stimulation index S.I. (indiv +/-  Standard deviation
1PGSf 0979

0.772


0.712


0.730


0.7380.794n.a.
1PGSf 0980

0.802


0.808


0.856


0.8230.794n.a.
1PGSf 0981

0.804


0.853


0.944


0.8670.794n.a.
1PGSf 0983

0.703


0.773


0.776


0.7470.794n.a.
210%Sf 0984

0.469


0.457


0.417


0.4480.5390.56+/-0.03
210%Sf 0.985

0.482


0.520


0.602


0.5350.5390.60+/-0.06
210%Sf 0986

0.482


0.520


0.602


0.5350.5390.67+/-0.08
210%Sf 0987

0.659


0.661


0.763


0.6940.5390.87+/-0.07
325%Sf 0989

0.485


0.542


0.568


0.5320.6170.67+/-0.05
325%Sf 0990

0.538


0.569


0.545


0.5510.6170.67+/-0.02
325%Sf 0991

0.612


0.535


0.679


0.6090.6170.77+/-0.09
325%Sf0992

0.695


0.686


0.951


0.7770.6170.98+/-0.19
450%Sf 0994

0.486


0.569


0.601


0.5790.608

0.73+/-0.03


450%Sf 0995

0.486


0.569


0.590


0.5480.6080.69+/-0.07
450%Sf 0996

0.493


0.545


0.606


0.5480.6080.69+/-0.07
450%Sf 0997

0.695


0.691


0.880


0.7550.608

0.95+/-0.14



 


BODY WEIGHTS





















































































































































































































   Bodyweight (g)Bodyweight (g) 
GroupsTesti itemAnimals No.Day 1Day 6Body weight gain (g)

1


PGSf 097920.421.61.2
1PGSf 098019.220.61.4
1PGSf 098121.022.41.4
1PGSf 098318.519.61.1
1 MEAN19.821.11.3
1 Standard-deviation1.11.20.1
210%Sf 098420.421.20.8
210%Sf 098519.319.50.2
210%Sf 098622.623.40.8
210%Sf 098720.720.90.2
2 MEAN20.821.30.5
2 Standard-deviation1.41.60.3
325%Sf 098920.422.62.2
325%Sf 099020.322.11.8
325%Sf 099120.021.41.4
325%Sf 099220.720.80.1
3 MEAN20.421.71.4
3 Standard-deviation0.30.80.9
450%Sf 099422.424.01.6
450%Sf 099519.523.43.9
450%Sf 099620.921.60.7
450%Sf 099721.022.81.8
4 MEAN21.023.02.0
4 Standard-deviation1.21.01.4

 


 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Theresults obtained, under these experimental conditions, enable to cnclude that the test item 1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile does not have to be classified as skin sensitizer, in accordance with the regulation EC No. 1272/2008 on classification, labelling and packaging of substances and mixtures. No signal word or hazard statement is required.

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