3-Quinolinecarboxylic acid, 7-chloro-1-cyclopropyl-1,4-dihydro-8-methyl-4-oxo-, ethyl ester

Endpoint summary

Description of key information

Skin sensitisation (in chemico): First key event (molecular initiating event): Data waiving. Preliminary to the study, in accordance with OECD Guideline 442C the solubility at 100 mM was assessed in all required solvents. It was not possible to obtain a solution homogeneous and stable enough for any of the solvents. Thus, the test item could not be tested in accordance with the DPRA method.

Skin sensitisation (in vitro): Second key event (activation of keratinocytes): Data waiving. Preliminary to the study, in accordance with OECD Guideline 442D the solubility was assessed in all required solvents and diffferent concentrations in culture medium. It was not possible to obtain a solution homogeneous and stable enough for any of the solvents and conditions. Thus, the test item could not be tested in accordance with the KeratinoSens assay.

Skin sensitisation (in vitro): Third key event (activation of dendritic cells): Data waiving. This test was not performed because the available method (OECD TG 442E) addressing this key event tends to produce false negatice results with substances with a Log Kow greater than 3.5, as it is the case with the substance under consideration.

Skin sensitisation (in vivo): Key study. Test method according to the OECD 442B Guideline with GLP. Under the experimental conditions the test item does not have to be classified as a skin sensitizer using the LLNA assay. The Stimulation Index (SI) calculated by individual approach was 0.96, 0.91 and 1.01 at concentrations of test item 20%, 40% and 60%, respectively.

Conclusion: As it was not possible to perform the in vitro/in chemico tests addressing all the three key events required in column 1 of REACH Annex VII, it was necessary to conduct an in vivo LLNA assay according to column 2 of REACH Annex VII. Based on the results of the in vivo assay, the test substance does not need to be classified as skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
Preliminary to the study, in accordance with OECD Guideline 442C the solubility at 100 mM was assessed in all required solvents. It was not possible to obtain a solution homogeneous and stable enough for any of the solvents. Thus, the test item cannot be tested in accordance with the DPRA method. See attached a summary of this preliminary solubility test.

Reference 2

Endpoint:

skin sensitisation: in vitro

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
Preliminary to the study, in accordance with OECD Guideline 442D the solubility of the test item was assessed in all required solvents and different concentrations in culture medium. It was not possible to obtain a solution homogeneous and stable enough for any of the solvents and conditions. Thus, the test item cannot be tested in accordance with the Keratino Sens assay. See attached a summary of this preliminary solubility test.

Reference 3

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin sensitisation study does not need to be conducted because the available in vitro test methods are not applicable for the substance and therefore an in vivo skin sensitisation study was conducted

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
According to column 2 of REACH Annex VII, the available in vitro test method addressing the key event activation of dentritic cells (h-CLAT method, OECD TG 442E) is not applicable for this substance because it tends to produce false negative results with substances with a Log Kow greater than 3.5, as it is the case with the substance under consideration.

Reason / purpose for cross-reference:

data waiving: supporting information

Reference 4

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 October 2020 - 19 October 2020

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: 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

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: 9-11 weeks old.
- Weight at study initiation: average weight 23.8-24.5 g (treated groups and control group)
- Housing: the animals were housed individually (to avoid any test item absorption by oral route) in a 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 5 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19ºC-25ºC.
- Humidity (%): 30%-70%.
- Air changes (per hr): at least 10 changes/hour
- Photoperiod (hrs dark / hrs light): 12 h light (7.00 to 19.00) / 12 h darkness

Vehicle:

propylene glycol

Concentration:

60%, 40% and 20%.

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS: 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 propylene glycol 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. Any signs of toxicity or excessive local irritation noted during this period were recorded. Ear thickness was recorded on day 1, day 3 and on day 6.

- Compound solubility: Not specified.
- Irritation: No sign of excessive irritation was noted in the animal treated at the tested concentration of 60%.
- Systemic toxicity: No mortality and no signs of systemic toxicity were noted.
- Ear thickness measurements: values were within the acceptable range.
- Erythema scores: no signs of erithema were observed.

MAIN STUDY
Groups of four mice were treated with the test item diluted at concentrations of 60%, 40 and 20% in propylene glycol (PG) based on the results of the pre-screen test. The 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, 3).
- Clinical observations: 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 weight: the bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).
- Ear thickness: 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 weighted in order to assess the irritation potential of the test item and the two lymph nodes per mouse were weighed.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Skin sensitisation. Local Lymph Node Assay:BrdU-ELISA
- Criteria used to consider a positive response: BrdU was measured by ELISA using a commercial kit (Roche Applied Science, Mannheim, Germany, Catalogue Number 11 647 229 001 - Batch No. 43507600). 100 µL of the suspension of lymph node cells (LNC) was added to the wells of a flat-bottom microplate 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.
The BrdU labelling index was defined as: BrdU labelling index = (ABSem - ABS blankem) - (ABSref - ABS blankref).
SI = Mean BrdU labelling index/mouse Treated Group / Mean BrdU labelling index/mouse Control Group.
The test item is regarded as a sensitiser if at least one concentration of the test item results is greater than 1.6 compared to control values.
However, the strength of the dose-response relationship, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result (SI value between 1.6 and 1.9) is declared positive. Any test item failing to produce a SI>1.6 is classified as a "non-sensitiser".
The EC1.6 value (theoretical concentration resulting in a SI value of 1.6) was detemined 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:
Groups of four mice were treated with the test item diluted at concentrations of 60%, 40 and 20% in propylene glycol (PG). The 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 of BrdU solution (10 mg/mL) was injected by intra-peritoneal route.
The Brdu solution was prepared by weighing 120.50 mg of 5-bromo-2'-deoxyuridine (SIGMA – Batch No. H1BH0686V) in a glass sample bottle and adding 12.05 mL of physiological saline. Then, the preparation was magnetically stirred during 20 minutes to obtain a colourless solution just before the treatment.
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 through 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 PBS (Ca2 +/Mg2+ - free) into a well of a multi-well 6. The optimised 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)

Positive control results:

EC1.6= 18.69%. The substance has to be classified in category 1 "Skin sensitisation".

Key result

Parameter:

SI

Value:

0.96

Variability:

SD = 0.18

Test group / Remarks:

20%

Key result

Parameter:

SI

Value:

0.91

Variability:

SD = 0.08

Test group / Remarks:

40%

Key result

Parameter:

SI

Value:

1.01

Variability:

SD = 0.14

Test group / Remarks:

60%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
The results of proliferation assay are given in tables 4 and 5 below.

DETAILS ON STIMULATION INDEX CALCULATION
No stimulation index of more than 1.6 was recorded whatever the tested concentration. The Stimulation Index (SI) calculated by individual approach was 1.01, 0.91 and 0.96 for the treated groups at 60%, 40%, 20%, respectively.

EC3 CALCULATION
The EC1.6 cannot be determined in this study.

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 60%, 40%, 20% and control animals during the test.

BODY WEIGHTS
Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness).
No increase in ear thickness and in ear weight was noted in animals treated at 20%, 40% and 60%.

Table 1. Preliminary screening test: Clinical observation, bodyweight and mortality data

 

CONCENTRATION %	ANIMAL	BODYWEIGHT (g)		DAY
		Day 1	Day 6	1	2	3	4	5	6
60%	Sf3202	21.8	20.8	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
Sf3202	0.20	0.19	0.21	28.4	5.0	No

 

The concentration of 60% was chosen as the highest concentration for the main study.

 

Table 2. Individual clinical observation and mortality data

 

Groups	Test item	Animals No	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6
1	PG	Sf 3161	0	0	0	0	0	0
		Sf3162 Sf3163	0	0	0	0	0	0
			0	0	0	0	0	0
		Sf 3164	0	0	0	0	0	0
2	20%	Sf 3165	0	0	0	0	0	0
		Sf3166 Sf3167	0	0	0	0	0	0
			0	0	0	0	0	0
		Sf 3168	0	0	0	0	0	0
3	40%	Sf 3170	0	0	0	0	0	0
		Sf3171 Sf3172	0	0	0	0	0	0
			0	0	0	0	0	0
		Sf 3173	0	0	0	0	0	0
4	60%	Sf 3175	0	0	0	0	0	0
		Sf3176 Sf3177	0	0	0	0	0	0
			0	0	0	0	0	0
		Sf 3178	0	0	0	0	0	0

0: no sign of erythema / no sign of systemic toxicity

PG: Propylene glycol

 

Table 3. Individual body weight and body weight gain

 

Groups	Test item	Animals No.	Bodyweight (g)		Body weight gain (g)
			Day 1	Day 6
1	PG	Sf 3161	26.6	28.1	1.5
		Sf 3162	23.1	23.8	0.7
		Sf 3163	21.9	20.9	-1.0
		Sf 3164	26.4	27.0	0.6
	MEAN		24.5	25.0	0.5
	Standard-deviation		2.4	3.3	1.0
2	20%	Sf 3165	26.4	25.7	-0.7
		Sf 3166	24.0	23.4	-0.6
		Sf 3167	22.3	23.5	1.2
		Sf 3168	22.6	23.2	0.6
	MEAN		23.8	24.0	0.1
	Standard-deviation		1.9	1.2	0.9
3	40%	Sf 3170	28.0	27.7	-0.3
		Sf 3171	22.9	22.6	-0.3
		Sf 3172	26.1	25.8	-0.3
		Sf 3173	20.0	20.6	0.6
	MEAN		24.3	24.2	-0.1
	Standard-deviation		3.5	3.2	0.5
4	60%	Sf 3175	25.8	25.8	0.0
		Sf 3176	23.7	23.3	-0.4
		Sf 3177	23.8	24.6	0.8
		Sf 3178	22.2	22.9	0.7
	MEAN		23.9	24.2	0.3
	Standard-deviation		1.5	1.3	0.6

PG: Propylene glycol

 

Table 4. BrdU index & Stimulation index per group and calculation of EC1.6

 

Groups	Test item	BrdU-index (mean*)	Stimulation Index SI (mean + standard deviation)	Result	EC1.6 value
1	PG	0.638	n.a	n.a	n.a.
2	20%	0.614	0.96 ± 0.18	negative	n.a.
3	40%	0.577	0.91 ± 0.08	negative
4	60%	0.645	1.01 ± 0.14	negative

n.a.: non applicable

*: mean: Σindividual value / 4

PG: Propylene glycol

 

Table 5. BrdU index & Stimulation index (individual data)

 

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	Sf3161	0.656	0.644	0.638	n.a
			0.651
			0.625
		Sf3162	0.657	0.659		n.a
			0.798
			0.523
		Sf3163	0.711	0.685		n.a
			0.730
			0.615
		Sf3164	0.562	0.563		n.a
			0.550
			0.577
2	20%	Sf3165	0.578	0.611	0.614	0.96 ± 0.04
			0.628
			0.626
		Sf3166	0.620	0.597		0.94 ± 0.07
			0.623
			0.548
		Sf3167	0.744	0.764		1.20 ± 0.08
			0.728
			0.820
		Sf3168	0.478	0.484		0.76 ± 0.03
			0.508
			0.466
3	40%	Sf3170	0.616	0.615	0.577	0.96 ± 0.07
			0.662
			0.567
		Sf3171	0.608	0.607		0.95 ± 0.01
			0.616
			0.597
		Sf3172	0.591	0.584		0.92 ± 0.02
			0.594
			0.567
		Sf3173	0.538	0.503		0.79 ± 0.10
			0.539
			0.431
4	60%	Sf3175	0.652	0.688	0.645	1.08 ± 0.05
			0.717
			0.696
		Sf3176	0.744	0.754		1.18 ± 0.03
			0.778
			0.741
		Sf3177	0.550	0.569		0.89 ± 0.03
			0.584
			0.572
		Sf3178	0.633	0.569		0.89 ± 0.09
			0.543
			0.530

*: mean: Σindividual value / 4

PG: Propylene glycol

  

Table 6. Individual Ear thickness and irritation level

 

Groups	Test item	Animals No.	Day 1 earthickness (mm)	Day 3 earthickness (mm)	Day 6 earthickness (mm)	Ear thickness increase D3/D1 (%)	Ear thickness increase D6/D1 (%)
1	PG	Sf 3161	0.21	0.21	0.20	0.0	-4.8
		Sf 3162	0.21	0.21	0.21	0.0	0.0
		Sf 3163	0.21	0.20	0.21	-4.8	0.0
		Sf 3164	0.21	0.21	0.21	0.0	0.0
	MEAN		0.21	0.21	0.21	-1.19	-1.19
	Standard-deviation		0.00	0.00	0.00	2.38	2.38
2	20%	Sf 3165	0.21	0.21	0.21	0.0	0.0
		Sf 3166	0.21	0.21	0.20	0.0	-4.8
		Sf 3167	0.20	0.21	0.20	5.0	0.0
		Sf 3168	0.21	0.21	0.21	0.0	0.0
	MEAN		0.21	0.21	0.21	1.3	-1.2
	Standard-deviation		0.00	0.00	0.01	2.5	2.4
3	40%	Sf 3170	0.21	0.21	0.22	0.0	4.8
		Sf 3171	0.21	0.21	0.19	0.0	-9.5
		Sf 3172	0.21	0.21	0.20	0.0	-4.8
		Sf 3173	0.21	0.21	0.20	0.0	-4.8
	MEAN		0.21	0.21	0.20	0.0	-3.6
	Standard-deviation		0.00	0.00	0.01	0.0	6.0
4	60%	Sf 3175	0.21	0.21	0.22	0.0	4.8
		Sf 3176	0.21	0.21	0.21	0.0	0.0
		Sf 3177	0.21	0.21	0.21	0.0	0.0
		Sf 3178	0.21	0.21	0.21	0.0	0.0
	MEAN		0.21	0.21	0.21	0.0	1.2
	Standard-deviation		0.00	0.00	0.01	0.0	2.4

PG: Propylene glycol

 

  

Table 7. Individual Ear biopsy weight and lymph node weight.

 

Groups	Test item	Animals No.	ear weight Day 6 (mg)	% of ear weight increased/group1	lymph nodes (mg)
1	PG	Sf 3161	27.3		8.2
		Sf 3162	27.5		5.1
		Sf 3163	27.4		5.6
		Sf 3164	26.9		5.1
	MEAN		27.3		6.0
	Standard-deviation		0.3		1.5
2	20%	Sf 3165	28.8	4.6	6.3
		Sf 3166	26.3		5.4
		Sf 3167	29.5		6.7
		Sf 3168	29.5		5.8
	MEAN		28.5		6.1
	Standard-deviation		1.5		0.6
3	40%	Sf 3170	29.4	-1.8	5.8
		Sf 3171	24.4		5.1
		Sf 3172	26.9		7.4
		Sf 3173	26.4		5.8
	MEAN		26.8		6.0
	Standard-deviation		2.1		1.0
4	60%	Sf 3175	30.5	6.8	7.4
		Sf 3176	28.5		7.8
		Sf 3177	28.4		6.0
		Sf 3178	29.1		5.7
	MEAN		29.1		6.7
	Standard-deviation		1.0		1.0

PG: Propylene glycol

 

Table 8. Summary of result – 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%	-1.2	4.6	No
3	40%	-3.6	-1.8	No
4	60%	1.2	6.8	No

#: O.E.C.D. criteria: % increase in ear thickness higher than 25%, score of erythema higher than 3.

PG: Propylene glycol

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Interpretation of results:

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

Conclusions:

The test item does not have to be classified as a skin sensitizer under the tested conditions in the LLNA assay (OECD 442B). The Stimulation Index (SI) calculated by individual approach were 0.96, 0.91 and 1.01 at concentrations of test item 20%, 40% and 60%, respectively.

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, following GLP. Firstly, a preliminary screening test was performed using one mouse treated with 25 μL of the test item diluted at 60% in propylene glycol (PG) for three consecutive days. No mortality or signs of systemic toxicity and no signs of excessive irritation were noted in the preliminary study. In the main test, three groups of four mouse CBA/J were treated for three consecutive days with 50 µL (25 µL per ear) of the test item diluted at concentrations of 60%, 40 and 20% in PG. A control group was treated with propylene glycol. 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. No mortality and no signs of systemic toxicity were noted in the test and control animals during the test. No increase in ear thickness and in ear weight was noted in animals treated at 20%, 40% and 60%. Therefore, the test item has to be considered as not excessively irritant at these concentrations.The Stimulation Index (SI) calculated by individual approach was 0.96, 0.91 and 1.01 at concentrations of test item 20%, 40% and 60%, respectively. Therefore, the EC1.6 could not be determined due to the absence of SI value higher than 1.6. Under these experimental conditions, the test item does not have to be classified as a skin sensitizer in accordance with the CLP Regulation (EC) no. 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data, the substance is not classified for skin sensitization according to CLP Regulation no. 1272/2008.