1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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

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

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.

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.

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".

DETAILS ON STIMULATION INDEX CALCULATION

Groups	Test item	Animal No.	BrdU-index (DO indiv)	BrdU-index (DO mean)	BrdU-index mean*	Stimulation index S.I. (indiv +/-  Standard deviation
1	PG	Sf 0979	0.772 0.712 0.730	0.738	0.794	n.a.
1	PG	Sf 0980	0.802 0.808 0.856	0.823	0.794	n.a.
1	PG	Sf 0981	0.804 0.853 0.944	0.867	0.794	n.a.
1	PG	Sf 0983	0.703 0.773 0.776	0.747	0.794	n.a.
2	10%	Sf 0984	0.469 0.457 0.417	0.448	0.539	0.56+/-0.03
2	10%	Sf 0.985	0.482 0.520 0.602	0.535	0.539	0.60+/-0.06
2	10%	Sf 0986	0.482 0.520 0.602	0.535	0.539	0.67+/-0.08
2	10%	Sf 0987	0.659 0.661 0.763	0.694	0.539	0.87+/-0.07
3	25%	Sf 0989	0.485 0.542 0.568	0.532	0.617	0.67+/-0.05
3	25%	Sf 0990	0.538 0.569 0.545	0.551	0.617	0.67+/-0.02
3	25%	Sf 0991	0.612 0.535 0.679	0.609	0.617	0.77+/-0.09
3	25%	Sf0992	0.695 0.686 0.951	0.777	0.617	0.98+/-0.19
4	50%	Sf 0994	0.486 0.569 0.601	0.579	0.608	0.73+/-0.03
4	50%	Sf 0995	0.486 0.569 0.590	0.548	0.608	0.69+/-0.07
4	50%	Sf 0996	0.493 0.545 0.606	0.548	0.608	0.69+/-0.07
4	50%	Sf 0997	0.695 0.691 0.880	0.755	0.608	0.95+/-0.14

 

BODY WEIGHTS

			Bodyweight (g)	Bodyweight (g)
Groups	Testi item	Animals No.	Day 1	Day 6	Body weight gain (g)
1	PG	Sf 0979	20.4	21.6	1.2
1	PG	Sf 0980	19.2	20.6	1.4
1	PG	Sf 0981	21.0	22.4	1.4
1	PG	Sf 0983	18.5	19.6	1.1
1		MEAN	19.8	21.1	1.3
1		Standard-deviation	1.1	1.2	0.1
2	10%	Sf 0984	20.4	21.2	0.8
2	10%	Sf 0985	19.3	19.5	0.2
2	10%	Sf 0986	22.6	23.4	0.8
2	10%	Sf 0987	20.7	20.9	0.2
2		MEAN	20.8	21.3	0.5
2		Standard-deviation	1.4	1.6	0.3
3	25%	Sf 0989	20.4	22.6	2.2
3	25%	Sf 0990	20.3	22.1	1.8
3	25%	Sf 0991	20.0	21.4	1.4
3	25%	Sf 0992	20.7	20.8	0.1
3		MEAN	20.4	21.7	1.4
3		Standard-deviation	0.3	0.8	0.9
4	50%	Sf 0994	22.4	24.0	1.6
4	50%	Sf 0995	19.5	23.4	3.9
4	50%	Sf 0996	20.9	21.6	0.7
4	50%	Sf 0997	21.0	22.8	1.8
4		MEAN	21.0	23.0	2.0
4		Standard-deviation	1.2	1.0	1.4

 

 

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.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The preliminary solubility study discarded the possibility of performing the study because of the test item solubility in the vehicle.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

not applicable

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
Placeholder - no text template available yet
Couldn't be performed because of the low solubility of the test item.

Run / experiment:

other: 1 run

Parameter:

other:

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

not determinable because of methodological limitations

Interpretation of results:

study cannot be used for classification

Conclusions:

The test item cannot be tested according to the DPRA method.

Reference 3

Endpoint:

skin sensitisation: in vitro

Remarks:

Human Cell Line Activation Test (h-CLAT)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 12 JUNE 2019 to 02 JULY 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 442E

Version / remarks:

OECD Guidelines for the Testing of Chemicals: OECD 442E; In Vitro Skin sensitisation: InVitro Skin Sensitisation Assays addressing the key Event on activation of dendritic cells on the Adverse Outcome Pathway from Skin Sensitisation. Annex I: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), June 2018.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

The substance has a low solubility and the test OECD 442C couldn't be performed. So, next step for to test skin sensitisation was h-CLAT test.

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
An alternative method has been developed for the evaluation of the skin sensitisation potential by measuring phenotypic changes, such as CD86 and CD54 expression on dendritic cells. The human leukemia cell line THP-1 is used as a surrogate for human myeloid dendritic cells, since these cells also show enchanced CD86 and/or CD 54 expression when treated with sensitisers.

The purpose of the Human Cell Line Activastion Test (h-CLAT) is to assess the skin sensitising potential of 1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile in an appropriate solvent (DMSO, saline or culture medium) when administered to THP-1 cells for 24 hours. The test item concentrations for the main experiment (h-CLAT) of 1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile are determined by cytotoxicity tests.

This human cell line activation test can be used as part of a testing battery (including e.g.
DPRA (Direct Peptide Reactivity Assay), ARE-Nrf2 luciferase test method) based on the
OECD adverse outcome pathway for the assessment of the skin sensitisation potential of
chemicals.

The technical proficiency of the h-CLAT with the OECD 442E guideline recommended
proficiency substances was demonstrated.

Positive control results:

Name: DNCB (2,4-dinitrochlorobenzene, CAS No.: 97-00-7) final concentration: 3 and 4 µg/mL, Purity ≥ 99%)
Solvent: DMSO

The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%, with one exception. The CD54 RFI value of the positive control (3.0 µg/mL DNCB) in the third h-CLAT run did not exceed the
positive criterion (CD54 ≥ 200%). However, this one exception is considered to be acceptable since the CD54 RFI value of the positive control (4.0 µg/mL DNCB) in the third h-CLAT run exceeded the positive criteria.

Acceptance criteria
In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50% in at least one
concentration of the two tested positive control concentrations.

Run / experiment:

other: Run 1

Parameter:

other: µg/mL

Remarks:

The concentration of the test item that could activate THP-1 cells. The maximum concentration is 3.91 µg/mL (limited by the solubility of the test item) under the test conditions study.

Value:

3.91

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

DCNB in DMSO.

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

The following acceptance criteria should be met when using the h-CLAT method:
• Cell viability of medium control and DMSO control should be more than 90%.
• In the solvent/vehicle control (i.e. DMSO), RFI values compared to the medium
control of both CD86 and CD54 should not exceed the positive criteria (CD86
≥ 150% and CD54 ≥ 200%).
• For both medium and solvent/vehicle controls (i.e. DMSO), the MFI ratio of CD86
and CD54 to isotype control should be > 105%.
• In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the
positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should
be > 50% in at least one concentration of the two tested positive control
concentrations.
• For the test chemical, the cell viability should be more than 50% in at least four tested
concentrations in each run.
Negative results are acceptable only for test items exhibiting a cell viability of < 90% at the
highest concentration tested (i.e. 1.2 × CV75). If the cell viability at 1.2 × CV75 is ≥ 90% the
negative result should be discarded. In such case it is recommended to try to refine the dose
selection by repeating the CV75 determination. It should be noted that when 5000 μg/mL in
saline (or medium or other solvents/vehicles), 1000 μg/mL in DMSO or the highest soluble
concentration is used as the maximal test concentration of a test chemical, a negative result is
acceptable even if the cell viability is > 90% (OECD 442E guideline).

Results of the first h-CLAT run for the Test Item 1-butyl-5-[(4-chlorophenyl)azo]-1,2-
dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile

Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	95.66
DMSO Control	-	100.0	100.0	94.80
Positive Control (DNCB)	3.0	551.0*	215.2*	86.24
4.0	697.1*	201.0*	85.23
Test Item	1.09	150.0	95.3	94.70
1.31	163.7	111.6	93.99
1.57	190.2	110.8	93.14
1.89	165.7	116.5	93.19
2.26	161.8	106.5	93.23
2.72	171.6	105.5	90.29
3.26	189.2	113.2	89.40
3.91	190.2	119.1	85.69

* RFI value of CD86 or CD54 fulfilled the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

 

Results of the second h-CLAT run for the Test Item 1-butyl-5-[(4-chlorophenyl)azo]-
1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile

Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	96.12
DMSO Control	-	100.0	100.0	95.31
Positive Control (DNCB)	3.0	264.5*	230.9*	87.77
4.0	442.1*	312.8*	82.31
Test Item	1.09	113.1	102.3	94.85
1.31	100.9	163.8*	94.33
1.57	126.2	133.6	91.94
1.89	117.8	128.3	92.58
2.26	123.4	168.4*	92.41
2.72	135.5	137.8	91.59
3.26	134.6	168.8*	90.42
3.91	128.0	163.8*	89.05

* RFI value of CD86 or CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

 

Results of the third h-CLAT run for the Test Item 1-butyl-5-[(4-chlorophenyl)azo]-1,2-
dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile

Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	90.47
DMSO Control	-	100.0	100.0	90.51
Positive Control (DNCB)	3.0	168.0#	183.0*	80.91
4.0	228.5*	205.1*	66.36
Test Item	1.09	104.7	102.1	90.65
1.31	114.1	103.5	88.74
1.57	130.1	96.5	89.14
1.89	116.8	97.9	88.38
2.26	125.4	104.4	87.60
2.72	141.4	139.4	87.56
3.26	153.5	135.0	85.93
3.91	151.6	134.0	84.30

* RFI value of CD86 or CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
# CD54 RFI value of the positive control (3.0 µg/mL DNCB) did not fulfil the positive criteria
(CD54 ≥ 200%)

Interpretation of results:

study cannot be used for classification

Conclusions:

The test item 1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-
oxonicotinonitrile with a log Pow of 5.181 did not activate THP-1 cells up to a concentration
of 3.91 µg/mL (limited by the solubility of the test item) under the test conditions of this
study. However, the negative result cannot be used in an assessment of skin sensitisation
potential because the log Pow of the test item is > 3.5 and according to the OECD test
Guideline 442E, negative results for test chemicals with a log Kow (Pow) > 3.5 should not be
considered.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

Despite the log Pow value of 5.2 (partition coefficient n-octanol/water, according to OECD 117), and the technical impossibility of performing the OECD 442C because of the solubility of the test item, was performed a non-conclusive "In vitro Skin Sensitisation Test - Human Cell Line Activation Test (h-CLAT) which conclusion was the negative conclusion to skin sensitising test but these results shouldn't be considered because the Log Pow > 3.5, according the OECD 442E criteria.

As the previous in vitro studies (in fact only h-CLAT study was completed), could not be used as a stand-alone or in a weight-of-evidence approach, it was considered as necessary to continue to an in vivo testing. As the LLNA (Local Lymph Node Assay) is the preferred and the recommended method when animal testing is necessary for the conclusion on the classification of the substance, was performed the study LLNA: BrdU ELISA according to OECD Guideline 442B with an appropriate vehicle. the outcome of the study was negative, i.e. that the substance is not sensitiser.