(6R,7R)-7-[[(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(triphenylmethoxy)imino]acetyl]amino]-3-[(E)-[(3'R)-1'-[(1,1-dimethylethoxy)carbonyl]-2-oxo-[1,3'-bipyrrolidin]-3-ylidene]methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid diphenylmethyl ester

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 March 2018 to 20 March 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

OECD Guidelines for Testing of Chemicals No. 429 Skin Sensitisation: Local Lymph Node Assay (22 July 2010)

Deviations:

yes

Remarks:

See "Any other information" for details

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

Commission Regulation (EC) No 440/2008 of 30 May 2008, B.42. Skin Sensitisation: Local Lymph Node Assay (Official Journal L 142, 31/05/2008) amended by Commission Regulation (EU) No 640/2012 of 6 July 2012

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

tert-butyl (3E,3'R)-3-{[(6R,7R)-7-[(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(triphenylmethoxy)imino]acetamido]-2-[(diphenylmethoxy)carbonyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl]methylidene}-2-oxo-[1,3'-bipyrrolidine]-1'-carboxylate

EC Number:

609-463-9

Cas Number:

376653-41-7

Molecular formula:

C57H54N8O8S2

IUPAC Name:

tert-butyl (3E,3'R)-3-{[(6R,7R)-7-[(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(triphenylmethoxy)imino]acetamido]-2-[(diphenylmethoxy)carbonyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl]methylidene}-2-oxo-[1,3'-bipyrrolidine]-1'-carboxylate

Test material form:

solid: particulate/powder

Details on test material:

Name: BAL0001026
CAS number: 376653-41-7
Batch/Lot number: 012
Appearance: Off-white powder
Purity: Content by mass balance: 84.9% w/w
Expiry date: 30 June 2020
Storage conditions: Under inert gas, protected from light and humidity (tight closed container), frozen (≤-15 °C)
Safety precautions: Enhanced safety precautions were applied considering the supplied safety datasheet to assure personnel health and safety.

Specific details on test material used for the study:

No further details specified in the study report.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Species and strain: CBA/CaOlaHsd mice
Source: Envigo, San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy
Hygienic level: SPF at arrival; standard housing conditions during the study
Justification of strain: On the basis of OECD Guideline, mice of CBA/Ca or CBA/J strain can be used. Females are used because the existing database is predominantly based on females.
Number of animals: 4 animals / group
Sex: Female, nulliparous, non-pregnant
Age of animals at starting: 12 weeks old (age-matched, within one week)
Body weight range at starting: 20.3 – 22.9 grams (The weight variation in animals in the study did not exceed ± 20 % of the mean weight.)
Acclimatisation time: 35 days
Note: In the Preliminary Experiment, mice of 9 weeks of age (18.8 - 19.6 grams) were used.

Animal health: Only healthy animals were used for the study. Health status was certified by the veterinarian.
Housing / Enrichment: Group caging / mice were provided with glass tunnel-tubes
Cage type: Type II. polypropylene / polycarbonate
Bedding: Bedding was available to animals during the study
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 17.1 – 26.9°C
Relative humidity: 22 - 78 %
Ventilation: 15-20 air exchanges/hour
The temperature and relative humidity were recorded twice every day during the acclimatisation and experimental phases.

Food and feeding
Animals received ssniff® SM Rat/Mouse – Breeding and Maintenance, 15 mm, autoclavable “Complete feed for Rats and Mice – Breeding and Maintenance" (Batch number: 382 24962, Expiry date: 30 April 2018, produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), and Gel diet Transport (Batch Number: 60172780020101, Expiry date: 05 October 2018, Scientific Animal Food & Engineering, Route de Saint Bris, 89290 Augy, France) ad libitum. The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Water supply
Animals received tap water from the municipal supply from 500 mL bottles, ad libitum. Water quality control analysis was performed once every three months and microbiological assessment was performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József Attila u. 36., Hungary). Copies of the relevant Certificates of Analysis are retained in the Archive at Citoxlab Hungary Ltd.

Bedding
Bedding of certified wood chips especially designed to keep animals in the best natural environment was provided for animals during the study. Lignocel 3/4-S Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH + Co.KG (D-73494 Rosenberg, Germany) was available to animals during the study. Certified nest building material was also provided for animals (ARBOCEL crinklets natural produced by J. Rettenmaier & Söhne GmbH + Co.KG).

Identification and randomisation
A unique number written on the tail with a permanent marker identified each animal. The animal number was assigned on the basis of Citoxlab Hungary Ltd.’s Master File. The cages were marked with identity cards with information including study code, cage number, and dose group, sex and individual animal number. The animals were randomised and allocated to the experimental groups. The randomisation was checked by computer software using the body weight to verify the homogeneity and variability between the groups.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

Negative (vehicle) control (DMF): - (% w/v test item concentration)
BAL0001026 50 % (w/v) in DMF: 50 (% w/v test item concentration)
BAL0001026 25 % (w/v) in DMF: 25 (% w/v test item concentration)
BAL0001026 10 % (w/v) in DMF: 10 (% w/v test item concentration)
Positive control (25% HCA in DMF): - (% w/v test item concentration)

No. of animals per dose:

4 animals per group (20 in total)

Details on study design:

Dose Selection and Justification of Dose Selection
The Preliminary Irritation/Toxicity Test was started according to the Study Plan on CBA/CaOlaHsd mice using two doses (2 animals/dose) at test item concentrations of 50 and 25 % (w/v) in DMF. The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and the radioactive proliferation assay was not performed.

The maximum concentration of test item in an acceptable solvent was established according to OECD guideline 429. Based on the observation of the solubility test, the maximum available concentration was 50 % (w/v).

In the Preliminary Irritation / Toxicity Test, all mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema and scored using Table 2 [4]. Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals.
During the Preliminary Irritation / Toxicity Test no mortality or clinical signs were observed. Test item precipitate / minimal amount of test item precipitate was observed on the ears of both animals in the 50 % (w/v) group between Day 1 and Day 4. Minimal amount of test item precipitate was observed on the ears of both animals in the 25 % (w/v) group.
A slight decrease was observed in body weight (>5% reduction) in 1 out of the 2 animals in the 50 % and in the 25 % (w/v) dose group also in the preliminary study. However, this fact was considered to have no toxicological significance and differences arise from individual variability. Average body weight loss of the groups were within 5%.
The draining auricular lymph nodes of the animals were visually examined: they were normal in both dose groups (subjective judgement by analogy with observations of former experiments).
Based on these observations, 50 % (w/v) dose was selected as top dose for the main test.

Topical application
During the study, animals were topically dosed with 25 µL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

PROLIFERATION ASSAY
Injection of Tritiated Thymidine (3HTdR)
On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS (phosphate buffered saline) containing approximately 20 μCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).

Removal and Preparation of Draining Auricular Lymph Nodes
Five hours (± 30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses).
The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels.
Once removed, the nodes of mice from each test group was pooled and collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.

Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). Pooled LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 °C. After centrifugation supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for each group of pooled lymph nodes.

Determination of Incorporated 3HTdR
After the final washing step, supernatants were removed. Pellets were gently agitated resuspended and 3 mL of 5 % (w/v) TCA solution was added to the tubes for precipitation of macromolecules.
After overnight (approximately 18 hours) incubation at 2-8 °C, precipitates were centrifuged (approximately 190 x g for 10 minutes at 4 °C), and supernatants were removed. Pellets were resuspended in 1 mL of 5 % (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).
The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5 % (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.

OBSERVATIONS
Clinical Observations
During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained.

Measurement of Body Weight
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

Positive control substance(s):

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

Statistics:

Not specified

Results and discussion

Positive control results:

The result of the positive control substance alpha-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. The positive control substance was examined at a concentration of 25 % in the relevant vehicle (DMF) using CBA/CaOlaHsd mice.
No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historical positive control data (stimulation index value of 5.0) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.
Furthermore, the DPN values observed for the vehicle and positive control substance in this experiment were in within the historical control range. Each treated and control group included 4 animals.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.6

Test group / Remarks:

50% (w/v) test item

Results 2

Key result

Parameter:

SI

Value:

0.8

Test group / Remarks:

25% (w/v) test item

Results 3

Key result

Parameter:

SI

Value:

0.8

Test group / Remarks:

10% (w/v) test item

Cellular proliferation data / Observations:

PROLIFERATION ASSAY
The appearance of the lymph nodes was normal in the negative control group and in the 50, 25 and 10 % (w/v) test item treated dose groups. Larger than normal lymph nodes were observed in the positive control group.

CLINICAL OBSERVATION
No mortality or signs of systemic toxicity were observed during the study. Test item precipitation / minimal amount of test item precipitation was observed on the ears in the 50 and 25 % (w/v) treatment groups from Day 1 to Day 5 in all animals and minimal amount of test item precipitation was observed on the ears in the 10 % (w/v) treatment group on Day 3 in 3 out of 4 animals.

BODY WEIGHT MEASUREMENT
No marked body weight losses (≥5%) were observed on the mean body weight changes in any groups; however, the body weight loss was between 5-10 % for 1 out of 4 animals in the negative control group and for 2 out of 4 animals in the 10 % (w/v) treatment group. This fact was considered to have no toxicological significance; such differences arise from individual variability.

INTERPRETATION OF OBSERVATIONS
The test item was powder, which was formulated in DMF. Since there were no confounding effects of irritation or systemic toxicity at the applied concentrations, the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay. The resulting stimulation indices observed under these exaggerated test conditions was considered to be good evidence that BAL0001026 is a non-sensitizer. The size of lymph nodes was in good correlation with this conclusion. Based on the observed results, the test item does not need classification according to the GHS or CLP.

Any other information on results incl. tables

Individual Body Weights for all Animals with Group Means

Animal Number	Identity Number	Test Group Name	Initial Body Weight (g)	Terminal Body Weight* (g)	Change# (%)
5627	1	Negative (vehicle) control in DMF	22.2	23.5	5.9
5633	2		21.6	22.0	1.9
5630	3		21.9	20.6	-5.9
5635	4		20.6	20.0	-2.9
		Mean	21.6	21.5	-0.2
5632	5	BAL00010226 50% (w/v) in DMF	22.5	22.1	-1.8
5639	6		21.6	21.4	-0.9
5640	7		21.3	21.5	0.9
5631	8		20.5	20.7	1.0
		Mean	21.5	21.4	-0.2
5638	9	BAL0001026 25% (w/v) in DMF	22.9	22.0	-3.9
5634	10		21.6	22.0	1.9
5628	11		21.2	22.5	6.1
5641	12		20.7	19.9	-3.9
		Mean	21.6	21.6	0.0
5637	13	BAL0001026 10% (w/v) in DMF	22.6	20.4	-9.7
5629	14		21.7	22.6	4.1
5642	15		21.5	20.3	-5.6
5636	16		20.9	21.0	0.5
		Mean	21.7	21.1	-2.8
5608	17	Positive control 25% (w/v) HCA in DMF	22.2	21.5	-3.2
5609	18		21.8	22.0	0.9
5607	19		21.7	21.3	-1.8
5610	20		20.3	20.4	0.5
		Mean	21.5	21.3	-0.9

Notes:

*: Terminal body weights were measured on Day 6.

#: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

 

DPM, DPN and Stimulation Index Values for all Groups

Test Group Name	Measured DPM / group	DPM	Number of lymph nodes	DPN	Stimulation Index
Background (5% (w/v) TCA)	32.5	-	-	-	-
Negative control (DMF)	5024	4991.5	8	623.9	1.0
BAL0001026 50% (w/v) in DMF	3221	3188.5	8	398.6	0.6
BAL0001026 25% (w/v) in DMF	3787	3754.5	8	469.3	0.8
BAL0001026 10% (w/v) in DMF	4067	4034.5	8	504.3	0.8
Positive control (25% (w/v) HCA in DMF)	25211	25178.5	8	3147.3	9.9

 

 

Summarized Clinical Observations

Group	Identity No.	CLINICAL OBSERVATIONS
		DAY 1	DAY 2	DAY 3	DAY 4	DAY 5	DAY 6
Negative control (DMF)	1	BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free	Symptom-free	Symptom-free	Symptom-free
	2	BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free	Symptom-free	Symptom-free	Symptom-free
	3	BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free	Symptom-free	Symptom-free	Symptom-free
	4	BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free	Symptom-free	Symptom-free	Symptom-free
BAL0001026 50% (w/v) in DMF	5	BT: symptom-free AT: symptom-free**	BT: symptom-free AT: symptom-free*	BT: symptom-free AT: symptom-free*	Symptom-free**	Symptom-free**	Symptom-free
	6	BT: symptom-free AT: symptom-free**	BT: symptom-free AT: symptom-free*	BT: symptom-free AT: symptom-free*	Symptom-free**	Symptom-free**	Symptom-free
	7	BT: symptom-free AT: symptom-free**	BT: symptom-free AT: symptom-free*	BT: symptom-free AT: symptom-free*	Symptom-free**	Symptom-free**	Symptom-free
	8	BT: symptom-free AT: symptom-free**	BT: symptom-free AT: symptom-free*	BT: symptom-free AT: symptom-free*	Symptom-free**	Symptom-free**	Symptom-free
BAL0001026 25% (w/v) in DMF	9	BT: symptom-free AT: symptom-free**	BT: symptom-free AT: symptom-free*	BT: symptom-free AT: symptom-free*	Symptom-free**	Symptom-free**	Symptom-free
	10	BT: symptom-free AT: symptom-free**	BT: symptom-free AT: symptom-free*	BT: symptom-free AT: symptom-free*	Symptom-free**	Symptom-free**	Symptom-free
	11	BT: symptom-free AT: symptom-free**	BT: symptom-free AT: symptom-free*	BT: symptom-free AT: symptom-free*	Symptom-free**	Symptom-free**	Symptom-free
	12	BT: symptom-free AT: symptom-free**	BT: symptom-free AT: symptom-free*	BT: symptom-free AT: symptom-free*	Symptom-free**	Symptom-free**	Symptom-free
BAL0001026 10% (w/v/) in DMF	13	BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free	Symptom-free	Symptom-free	Symptom-free
	14	BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free**	Symptom-free	Symptom-free	Symptom-free
	15	BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free**	Symptom-free	Symptom-free	Symptom-free
	16	BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free**	Symptom-free	Symptom-free	Symptom-free
Positive control (25% (w/v) HCA in DMF)	17	BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free	Symptom-free	Symptom-free	Symptom-free
	18	BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free	Symptom-free	Symptom-free	Symptom-free
	19	BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free	Symptom-free	Symptom-free	Symptom-free
	20	BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free	Symptom-free	Symptom-free	Symptom-free

Notes:

1. BT: before treatment, AT: after treatment

2. *: Test Item precipitate

3. **: Minimal amount of test item precipitate

 

Results of the Preliminary Irritation / Toxicity Test

 

Individual Body Weights for all Animals with Group Means (Preliminary Irritation/Toxicity Test)

Animal Number	Identity Number	Test Group Name	Initial Body Weight (g)	Terminal Body Weight* (g)	Change# (%)
5478	1	50% (w/v)	19.4	18.4	-5.2
5481	2	50% (w/v)	18.8	19.6	4.3
		Mean	19.1	19.0	-0.5
5480	3	25% (w/v)	19.4	19.2	-1.0
5479	4	25% (w/v)	19.6	18.3	-6.6
		Mean	19.5	18.8	-3.8

Notes:

1. *: Terminal body weights were measured on Day 6

2. #: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

 

Individual Ear Thickness for all Animals (Preliminary Irritation/Toxicity Test)

Animal Number	Identity Number	Test Group Name	Ear Thickness on Day 1 (mm)		Ear Thickness on Day 3 (mm)		Ear Thickness on Day 6 (mm)		Biopsy weight* on Day 6 (mg)
			Right	Left	Right	Left	Right	Left
5478	1	50% (w/v)	0.20	0.21	0.21	0.21	0.21	0.21	14.67
5481	2	50% (w/v)	0.21	0.21	0.22	0.21	0.21	0.22	15.24
5480	3	25% (w/v)	0.20	0.21	0.21	0.21	0.20	0.21	15.08
5479	4	25% (w/v)	0.20	0.21	0.21	0.22	0.21	0.21	14.14

Note:

1. *: Historical control range: 11.92-22.53 mg. Positive response is over 28.16 mg (≥25%).

 

Summarized Clinical Observations (Preliminary Irritation/Toxicity Test)

Period	Group	Identity No.	Animal No.	Clinical observations
DAY 1	50% (w/v)	1	5478	Before treatment: symptom-free, ES: 0 After treatment: symptom-free**, ES: 0
		2	5481	Before treatment: symptom-free, ES: 0 After treatment: symptom-free**, ES: 0
	25% (w/v)	3	5480	Before treatment: symptom-free, ES: 0 After treatment: symptom-free**, ES: 0
		4	5479	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
DAY 2	50% (w/v)	1	5478	Before treatment: symptom-free, ES: 0 After treatment: symptom-free*, ES: 0
		2	5481	Before treatment: symptom-free, ES: 0 After treatment: symptom-free*, ES: 0
	25% (w/v)	3	5480	Before treatment: symptom-free, ES: 0 After treatment: symptom-free**, ES: 0
		4	5479	Before treatment: symptom-free, ES: 0 After treatment: symptom-free**, ES: 0
DAY 3	50% (w/v)	1	5478	Before treatment: symptom-free, ES: 0 After treatment: symptom-free*, ES: 0
		2	5481	Before treatment: symptom-free, ES: 0 After treatment: symptom-free*, ES: 0
	25% (w/v)	3	5480	Before treatment: symptom-free, ES: 0 After treatment: symptom-free**, ES: 0
		4	5479	Before treatment: symptom-free, ES: 0 After treatment: symptom-free**, ES: 0
DAY 4	50% (w/v)	1	5478	Symptom-free**, ES: 0
		2	5481	Symptom-free**, ES: 0
	25% (w/v)	3	5480	Symptom-free**, ES: 0
		4	5479	Symptom-free, ES: 0
DAY 5	50% (w/v)	1	5478	Symptom-free, ES: 0
		2	5481	Symptom-free, ES: 0
	25% (w/v)	3	5480	Symptom-free, ES: 0
		4	5479	Symptom-free, ES: 0
DAY 6	50% (w/v)	1	5478	Symptom-free, ES: 0
		2	5481	Symptom-free, ES: 0
	25% (w/v)	3	5480	Symptom-free, ES: 0
		4	5479	Symptom-free, ES: 0

Notes:

1. The clinical observation of animas on the first day was performed simultaneously with the body weight measurements.

2. ES: Erythema score

3. *: Test Item precipitate, **: Minimal amount of Test Item precipitate

 

Historical Control Data

Historical Control Data of the Positive and Negative Controls for CBA/CaOlaHsd mice (2014-2018)

CBA/CaOlaHsd mice
	Vehicles
	Acetone:Olive oil 4:1 (AOO)			PE9200 in water (1%Plu)
	DPN values		SI value	DPN values		SI value
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
Average	472.7	3851.3	9.0	198.7	1988.1	11.2
Range: min	35.8	890.3	3.3	23.0	154.0	3.0
max	1990.1	10336.0	20.2	680.8	6755.8	33.6
Number of cases	92	88	86	234	226	218
	Vehicles
	N,N-Dimethylformamide (DMF)			Dimethyl sulfoxide (DMSO)
	DPN values		SI value	DPN values		SI values
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
Average	256.1	2738.9	11.3	466.0	3014.4	7.2
Range: min	62.0	1201.3	4.9	218.3	1461.3	3.1
max	649.6	5817.9	21.3	934.6	4877.5	14.5
Number of cases	68	68	68	22	22	21
	Vehicles
	Propylene glycol (PG)			Methyl ethyl ketone (MEK)
	DPN values		SI value	DPN values		SI values
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
Average	245.1	2278.6	9.4	264.5	4129.5	16.9
Range: min	63.3	817.3	5.8	80.5	1562.5	8.8
max	506.0	4978.0	14.4	516.2	8682.5	36.3
Number of cases	18	18	18	18	19	19
HCA 25% = alpha-Hexylcinnamaldhyde 26% (w/v)
SI (Stimulation Index) = DPN of treated group divided by DPN of the appropriate control group.
DPN (Disintegrations Per Node) = DPM (Disintegrations Per Minute) divided by the number of lymph nodes.
In case of individual approach, SI values were calculated from the mean DPN values of the group.

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, under the conditions of the present assay, BAL0001026, tested in a suitable vehicle, did not show a sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.
No classification/labelling is triggered according to Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 7) 2017.

Executive summary:

The object of this study was to determine the skin sensitisation potential of BAL0001026 following dermal exposure in mice. The study was being performed with vertebrate animals as the applied regulatory in vitro alternative tests* showed inconclusive/equivocal results. Therefore, an in vivo study was being run to provide reliable information about the skin sensitisation potential of the test item for regulatory acceptance.

 

*IMPORTANT NOTE: With Commission Regulation (EU) 2016/1688 adopted on the 20th September 2016, the testing strategy for the assessment of the skin sensitization potential of a new substance now includes mandatory in vitro testing before any in vivo testing can be carried out, if the in vitro tests are technically feasible. This applies to all substances to be tested for EU REACH and CLP, but not for other regulatory purposes.

 

Before the start of this in vivo study, the Sponsor confirmed that existing data was not sufficient for the labelling or for the specific regulatory purpose for Skin Sensitisation.

 

Based on the results of the Preliminary Compatibility Test, the test item characteristics, its usage and on the recommendations of the OECD Guideline, the best vehicle for the test item was N,N-dimethylformamide (DMF). The 50 % (w/v) formulation was the highest concentration suitable for the test. The 50% (w/v) formulation appeared, by visual examination, to be a suspension and the 25 and 10 % (w/v) formulations appeared to be solutions.

 

A Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose): 50 and 25 % (w/v) in DMF and based on the results, 50 % (w/v) was selected as top dose for the main test.

 

In the main assay, twenty female CBA/CaOlaHsd mice were allocated to five groups, each group comprised four animals:

-groups (three) of animals received BAL0001026 (formulated in DMF) at either 50, 25 or 10 % (w/v),

-a negative control group received the vehicle (DMF) only,

-a positive control group received 25 % (w/v) HCA (dissolved in DMF). (To minimise animal use, the positive control was part of a parallel study (17/219-037E).

 

The test item solutions were applied to the dorsal surface of the ears of the experimental animals (25 µL/ear) for three consecutive days (Days 1, 2 and 3) and then maintained on study for an additional 3 days. Cell proliferation in the (local) lymph nodes was assessed by measuring disintegrations per minutes after the incorporation of tritiated methyl thymidine (3HTdR) into the lymph nodes and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.

 

There was no mortality or signs of systemic toxicity observed during the study. Test item precipitation / minimal amount of test item precipitation was observed on the ears in the 50 and 25 % (w/v) treatment groups from Day 1 to Day 5 in all animals. In addition, a minimal amount of test item precipitation was observed on the ears in the 10 % (w/v) treatment group on Day 3 in 3 out of the 4 animals. No marked group mean body weight losses (≥5%) were observed in any groups.

 

The SI values were 0.6, 0.8 and 0.8 at concentrations of 50, 25 and 10 % (w/v), respectively. The SI value for the positive control substancea-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was 5.0 therefore demonstrating the appropriate performance of the assay. The lymphoproliferative response of the HCA was in line with historical control data for the positive control, therefore confirming the validity of the assay.

 

In conclusion, under the conditions of the present assay, BAL0001026, tested in DMF, did not show any sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.

 

No classification/labelling is triggered according to Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 7) 2017.