3-Quinolinecarboxylic acid, 7-[6-(benzoylmethylamino)-5-methyl-3-pyridinyl]-1-cyclopropyl-1,4-dihydro-8-methyl-4-oxo-, ethyl ester

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09.12.2020 - 22.12.2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin sensitisation: Local Lymph Node Assay: BrdU-ELISA or –FCM)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.51 (Skin Sensitisation: Local Lymph Node Assay: BrDU-ELISA)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Test material information

Constituent 1

Reference substance name:

3-Quinolinecarboxylic acid, 7-[6-(benzoylmethylamino)-5-methyl-3-pyridinyl]-1-cyclopropyl-1,4-dihydro-8-methyl-4-oxo-, ethyl ester

EC Number:

610-204-7

Cas Number:

446299-90-7

Molecular formula:

C30H29N3O4

IUPAC Name:

3-Quinolinecarboxylic acid, 7-[6-(benzoylmethylamino)-5-methyl-3-pyridinyl]-1-cyclopropyl-1,4-dihydro-8-methyl-4-oxo-, ethyl ester

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Remarks:

CBA/JRj

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Elevage Janvier Labs (F-53941Le Genest Saint Isle).
- Females (if applicable) nulliparous and non-pregnant: yes.
- Age at study initiation: 8-9 weeks old.
- Weight at study initiation: 20.2-25.0 g (treated groups and control group)
- Housing: The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: Teklad Global 16% Protein Rodent Diet (ENVIGO 2016) ad libitum
- Water: tap water from public distribution system ad libitum. Microbiological and chemical analyses of the water were carried out once every six months by Bureau Veritas - Eurofins (FRANCE).
- Acclimation period: at least five days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25ºC
- Humidity (%): 30-70%
- Air changes (per hr): at least ten changes per hour
- Photoperiod (hrs dark / hrs light): 12 h light (7.00 to 19.00) / 12 h darkness

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

60%, 40% and 20%.

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
As no information was available regarding irritant potential or systemic toxicity of the test item in the mouse, a preliminary screening test was performed using one mouse with the highest technically possible concentration. The mouse was treated by daily application of 25 μL of the test item diluted at 60% in PG to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed daily from day 1 to day 6. Ear thickness was recorded on day 1, day 3 and on day 6. The bodyweight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6.
- Compound solubility: Not specified
- Irritation: No sign of excessive irritation was noted at the tested concentration of 60%.
- Systemic toxicity: No mortality was noted at the tested concentrations of 60%.
- Ear thickness measurements: values were within the acceptable range.
- Erythema scores: 0 (no sign of erythema)

MAIN STUDY:
-Three groups of four mice each were treated with the test item diluted at 60%, 40% and 20% in PG. A further group of four mice received the vehicle alone. All four groups of mice were treated by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3).
-Clinical Observations and mortality: All animals were observed daily on Days 1, 2, 3, 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
-Body weights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination)
-Ear thickness measurements and recording of local reactions: On day 1 and on day 3 (before application) as well as on day 6 (after sacrifice) of each experiment, the thickness of the right ear of each animal of the vehicle control and treated groups was measured by a micrometer. Furthermore, on day 6, punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and weighed in order to assess the irritation potential of the test item and the two lymph nodes per mouse were weighed.
- Stimulation index (SI) determination: BrdU was measured by ELISA using a commercial kit. Briefly, 100 μL of the LNC suspension was added to the wells of a flat-bottom microplate at least in triplicate. After fixation and denaturation of the LNC, anti-BrdU antibody was added to each well and allowed to react. Subsequently the anti-BrdU antibody was removed by washing and the substrate solution was then added and allowed to produce chromogen. After 5 to 30 min, 30 μL of 1 M H2SO4 was added in each well, then shaken for one minute. Absorbance at 450 nm with a reference wavelength of 690 nm was then measured.

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: Results for each treatment group are expressed as the mean SI. The SI was derived by dividing the mean BrdU labelling index/mouse within each test group by the mean BrdU labelling index for the control group.
The BrdU labelling index was defined as:
BrdU labelling index = (ABSem – ABS blankem) – (ABSref – ABS blankref)
The test item will be regarded as a sensitiser if at least one concentration of the test item results is greater than 1.6 compared to control values.
-Determination of the EC1.6 value (theoretical concentration resulting in a SI value of 1.6): It was determined by linear interpolation of points on the dose-response curve, immediately above and below the 1.6-fold threshold. The equation used for calculation of EC1.6 was:
EC1.6 = c + [(1.6 – d) / (b – d)] x (a – c)
Legend: a = the lowest concentration giving stimulation index > 1.6
b = the actual stimulation index caused by a
c = the highest concentration failing to produce a stimulation index of 1.6
d = the actual stimulation index caused by c

According to Regulation (EC) No. 286/2011, the positive test item will be classified in subcategory 1A or 1B in accordance with:
If the EC value ≤ 2, the test item will be classified in "sub-category 1A".
If the EC value > 2, the test item will be classified in "sub-category 1B".

TREATMENT PREPARATION AND ADMINISTRATION
-Three groups of four mice each were treated with the test item diluted at 60%, 40% and 20% in PG. A further group of four mice received the vehicle alone. All four groups of mice were treated by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of four mice received the vehicle alone in the same manner.
- On day 5, 0.5 mL (5 mg/mouse) of BrdU (10 mg/mL) solution was injected by intra-peritoneal route. The BrdU solution was prepared by weighing 177.70 mg of 5-bromo-2'-deoxyuridine in a glass sample bottle and adding 17.77 mL of physiological saline. The preparation was then stirred by ultraturrax for 34 minutes just before the administration to obtain a colourless solution.
-On day 6 (end of the test), the animals were euthanized with sodium pentobarbital (Dolethal®).
The draining auricular lymph nodes from the four mice were excised.
-From each mouse, a single-cell suspension of lymph node cells (LNC) excised bilaterally was prepared by gentle mechanical disaggregation through a disposable plastic pestle to crush the lymph nodes followed by passage through a #70 nylon mesh in 15 mL of DPBS (Ca2+ / Mg2+ - free) into a well of a multi-well 6. The optimized volume was based on achieving a mean absorbance of the negative control group within 0.1- 0.2.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:

EC1.6= 18.69%. Under the experimental conditions tested, the positive control substance has to be classified in category 1 “Skin sensitisation”, in accordance with the Regulation (EC) No. 1272/2008.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.86

Test group / Remarks:

20%

Results 2

Key result

Parameter:

SI

Value:

0.99

Test group / Remarks:

40%

Results 3

Key result

Parameter:

SI

Value:

0.96

Test group / Remarks:

60%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
No increase in ear thickness and in ear weight was noted in animals treated at 20%, 40%, 60% respectively.

DETAILS ON STIMULATION INDEX CALCULATION
No stimulation index higher than 1.6 was recorded whatever the tested concentration. The Stimulation Index (SI) calculated by individual approach was 0.86, 0.99 and 0.96 for the treated groups at 20%, 40%, 60%, respectively.

EC3 CALCULATION
The EC1.6 cannot be determined due to the absence of SI value higher than 1.6.

CLINICAL OBSERVATIONS:
No mortality was noted in the test and control animals during the test.
No signs of systemic toxicity were noted in the test animals treated at 20%, 40%, 60% and control animals during the test.

BODY WEIGHTS
No change in body weight was noted for all treated groups versus control group.

Any other information on results incl. tables

Table 1. Results of the preliminary test at 60% concentration of the test item.

Concentration %	Animal	Bodyweight (g)		Day
		Day 1	Day 6	1	2	3	4	5	6
60%	Sf3214	21.0	22.5	0	0	0	0	0	0
				N.t.R.	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R

0: no sign of erythema, N.t.R.: Nothing to report (no sign of systemic toxicity)

ANIMAL	Ear thickness (mm) on day 1	Ear thickness (mm) on day 3	Ear thickness (mm) on day 6	Ear weight (mg) on day 6	Weight Lymph nodes (mg)	Excessive irritation
Sf3214	0.21	0.21	0.22	34.4	11.0	No

 

Table 2. Clinical observations and mortality data for test and control animals

1	PG	Sf 3218 Sf 3219 Sf 3220 Sf 3221	0	0	0	0	0	0
			0	0	0	0	0	0
			0	0	0	0	0	0
			0	0	0	0	0	0
2	20%	Sf 3243 Sf 3244 Sf 3245 Sf 3246	0	0	0	0	0	0
			0	0	0	0	0	0
			0	0	0	0	0	0
			0	0	0	0	0	0
3	40%	Sf 3248 Sf 3249 Sf 3250 Sf 3251	0	0	0	0	0	0
			0	0	0	0	0	0
			0	0	0	0	0	0
			0	0	0	0	0	0
4	60%	Sf 3253 Sf 3254 Sf 3255 Sf 3256	0	0	0	0	0	0
			0	0	0	0	0	0
			0	0	0	0	0	0
			0	0	0	0	0	0

0: no sign of erythema

Groups	Test item	AnimalsNo.	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6
1	PG	Sf 3218 Sf 3219 Sf 3220 Sf 3221	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
			N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
			N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
			N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
2	20%	Sf 3243 Sf 3244 Sf 3245 Sf 3246	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
			N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
			N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
			N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
3	40%	Sf 3248 Sf 3249 Sf 3250 Sf 3251	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
			N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
			N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
			N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
4	60%	Sf 3253 Sf 3254 Sf 3255 Sf 3256	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
			N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
			N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
			N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R

N.t.R.: Nothing to report (no sign of systemic toxicity)

 

Table 3. Individual bodyweights and bodyweight changes for test and control animals

Groups	Test item	Animals No.	Bodyweight (g)		Body weight gain (g)
			Day 1	Day 6
1	PG	Sf 3218	23.0	23.2	0.2
		Sf 3219	20.9	21.1	0.2
		Sf 3220	21.8	22.8	1.0
		Sf 3221	23.7	23.5	-0.2
	MEAN		22.4	22.7	0.3
	Standard-deviation		1.2	1.1	0.5
2	20%	Sf 3243	25.0	25.0	0.0
		Sf 3244	20.8	22.3	1.5
		Sf 3245	21.5	22.5	1.0
		Sf 3246	19.5	20.1	0.6
	MEAN		21.7	22.5	0.8
	Standard-deviation		2.4	2.0	0.6
3	40%	Sf 3248	22.0	22.1	0.1
		Sf 3249	22.4	23.3	0.9
		Sf 3250	22.2	22.5	0.3
		Sf 3251	22.2	22.7	0.5
	MEAN		22.2	22.7	0.5
	Standard-deviation		0.2	0.5	0.3
4	60%	Sf 3253	20.6	23.0	2.4
		Sf 3254	21.5	22.1	0.6
		Sf 3255	23.4	24.0	0.6
		Sf 3256	20.2	19.1	-1.1
	MEAN		21.4	22.1	0.6
	Standard-deviation		1.4	2.1	1.4

 

Table 4. Results of the proliferation assay (1/2)

Groups	Test item	BrdU-index (mean*)	Stimulation Index SI (mean + standard deviation)	Result	EC1.6 value
1	PG	0.621	n.a	n.a	n.a.
2	20%	0.531	0.86 ± 0.12	negative	n.a.
3	40%	0.617	0.99 ± 0.08	negative
4	60%	0.598	0.96 ± 0.19	negative

 

Table 5. Results of the proliferation assay (2/2)

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 3218	0.633 0.599 0.572	0.602	0.621	n.a
		Sf 3219	0.749 0.939 0.440	0.710		n.a
		Sf 3220	0.715 0.424 0.586	0.575		n.a
		Sf 3221	0.639 0.640 0.504	0.595		n.a
2	20%	Sf 3243	0.313 0.588 0.653	0.518	0.531	0.83 ± 0.29
		Sf 3244	0.728 0.559 0.510	0.599		0.97 ± 0.18
		Sf 3245	0.477 0.747 0.512	0.579		0.93 ± 0.24
		Sf 3246	0.516 0.356 0.418	0.429		0.69 ± 0.13
3	40%	Sf 3248	0.534 0.485 0.879	0.633	0.617	1.02 ± 0.35
		Sf 3249	0.623 0.570 0.647	0.614		0.99 ± 0.06
		Sf 3250	0.681 0.656 0.665	0.669		1.08 ± 0.02
		Sf 3251	0.674 0.423 0.556	0.551		0.89 ± 0.20
4	60%	Sf 3253	0.465 0.690 0.475	0.544	0.598	0.88 ± 0.20
		Sf 3254	0.342 0.728 0.396	0.490		0.79 ± 0.34
		Sf 3255	0.568 0.623 0.587	0.594		0.96 ± 0.05
		Sf 3256	0.708 0.808 0.775	0.763		1.23 ± 0.08

 

Table 6. Individual ear thickness and irritation level

Groups	Test item	Animals No.		Day 1 ear thickness  (mm)	Day 3 ear thickness  (mm)	Day 6 ear thickness (mm)	Ear thickness increase D3/D1 (%)	Ear thickness increase D6/D1 (%)
1	PG	Sf	3218	0.21	0.20	0.20	-4.8	-4.8
		Sf	3219	0.20	0.21	0.19	5.0	-5.0
		Sf	3220	0.20	0.21	0.21	5.0	5.0
		Sf	3221	0.21	0.21	0.21	0.0	0.0
	MEAN			0.21	0.21	0.20	1.31	-1.19
	Standard-deviation			0.01	0.00	0.01	4.68	4.73
2	20%	Sf	3243	0.21	0.21	0.18	0.0	-14.3
		Sf	3244	0.20	0.21	0.19	5.0	-5.0
		Sf	3245	0.19	0.21	0.19	10.5	0.0
		Sf	3246	0.21	0.20	0.21	-4.8	0.0
	MEAN			0.20	0.21	0.19	2.7	-4.8
	Standard-deviation			0.01	0.00	0.01	6.6	6.7
3	40%	Sf	3248	0.21	0.21	0.21	0.0	0.0
		Sf	3249	0.20	0.21	0.19	5.0	-5.0
		Sf	3250	0.21	0.21	0.19	0.0	-9.5
		Sf	3251	0.21	0.21	0.19	0.0	-9.5
	MEAN			0.21	0.21	0.20	1.3	-6.0
	Standard-deviation			0.00	0.00	0.01	2.5	4.5
4	60%	Sf	3253	0.21	0.20	0.21	-4.8	0.0
		Sf	3254	0.21	0.20	0.19	-4.8	-9.5
		Sf	3255	0.21	0.20	0.21	-4.8	0.0
		Sf	3256	0.21	0.20	0.19	-4.8	-9.5
	MEAN			0.21	0.20	0.20	-4.8	-4.8
	Standard-deviation			0.00	0.00	0.01	0.0	5.5

 

Table 7. Individual ear biopsy and lymph node weights

Groups	Test item	Animals No.		ear weight Day 6 (mg)	% of ear weight increased/group1	lymph nodes  (mg)
1	PG	Sf	3218	29.4		4.2
		Sf	3219	25.8		5.1
		Sf	3220	26.8		4.4
		Sf	3221	25.2		3.3
	MEAN			26.8		4.3
	Standard-deviation			1.9		0.7
2	20%	Sf	3243	26.0	1.8	3.3
		Sf	3244	27.5		4.9
		Sf	3245	26.9		4.1
		Sf	3246	28.7		3.8
	MEAN			27.3		4.0
	Standard-deviation			1.1		0.7
3	40%	Sf	3248	25.6	0.4	5.0
		Sf	3249	26.5		5.4
		Sf	3250	28.7		4.5
		Sf	3251	26.8		4.5
	MEAN			26.9		4.9
	Standard-deviation			1.3		0.4
4	60%	Sf	3253	27.0	4.2	5.3
		Sf	3254	28.0		5.0
		Sf	3255	28.2		6.5
		Sf	3256	28.5		5.8
	MEAN			27.9		5.7
	Standard-deviation			0.7		0.7

 

Table 8. Summary of results- skin irritation.

Groups	Test item	Ear thickness increase D6/D1 (%)	Biopsy ear weight Increase (%)	Excessive irritation #
1	PG	-1.2	n.a	No
2	20%	-4.8	1.8	No
3	40%	-6.0	0.4	No
4	60%	-4.8	4.2	No

Applicant's summary and conclusion

Interpretation of results:

other: Not classified (Regulation EC No. 1272/2008)

Conclusions:

The Stimulation Index (SI) calculated by individual approach in the in vivo LLNA assay was 0.86, 0.99 and 0.96 for the treated groups at 20%, 40%, 60%, respectively. Therefore, the test item is classified as non-sensitiser.

Executive summary:

The skin sensitisation potential of the test item was tested in the LLNA assay according to OECD 442B and E.U. B.51 method, under GLP conditions. A preliminary screening test was performed using one mouse (CBA:J) that was treated by daily application of 25 μL of the test item diluted at 60% in propylene glycol to the dorsal surface of each ear for three consecutive days. Neither mortality nor sign of excessive irritation was noted at the tested concentration of 60%. Therefore, 60% was chosen as the highest concentration for the main study. Three groups of four female mice each were treated with the test item diluted at 60%, 40% and 20% in propylene glycol (vehicle) and a fourth one of four mice received the vehicle alone.  On day 5, 0.5 mL of BrdU solution (10mg/mL) was injected by intraperitoneal route. On day 6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by measurement of BrdU content in DNA of lymphocyte using an ELISA kit. A positive control study using alpha-hexylcinnamaldehyde was also performed, leading to a EC1.6 value of 18.69% (skin sensitiser, category 1).
No mortality was noted in the test and control animals during the test. No signs of systemic toxicity were noted in the test animals treated at 60%, 40%, 20% and control animals during the test and no statistical significant change in body weight was noted for all treated groups versus control group. No stimulation index higher than 1.6 was recorded whatever the tested concentrations.
The Stimulation Index (SI) calculated by individual approach was 0.96, 0.99 and 0.86 for the treated groups at 60%, 40%, 20%, respectively. No increase in ear thickness and in ear weight was noted in animals treated with the test item. Therefore, the test item has to be considered as not excessively irritant at these concentrations and the test item does not have to be classified as a skin sensitiser, in accordance with the Regulation EC No. 1272/2008.