Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

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 guidelineopen allclose all
Qualifier:
according to
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
Qualifier:
according to
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. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Name: BAL0001024
CAS number: 376653-37-1
Batch/Lot number: 013
Appearance: Brown powder
Purity: 63.1% (content by qNMR)
Expiry date: 31 May 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 – 23.0 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 (19.5 – 20.0 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 - 76 %
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)
BAL0001024 25 % (w/v) in DMF: 25 (% w/v test item concentration)
BAL0001024 10 % (w/v) in DMF: 10 (% w/v test item concentration)
BAL0001024 5 % (w/v) in DMF: 5 (% 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 four doses (2 animals/dose) at test item concentrations of 100, 50, 26 and 10 % (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 100 % (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. 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.
Alopecia around the ears was present in the 100% (w/v) group from Day 5 (2 out of 2 animals).Test item precipitate / minimal amount of test item precipitate was observed on the ears of all animals in the 100% (w/v) dose group from Day 1 up to Day 5. Test item precipitate was observed on the ears of all animals in the 50% (w/v) dose group from Day 1 up to Day 3.
Decrease in the body weight was observed (>5% reduction) in 1 out of the 2 animals in the 50% dose group, and this change moved the group average for this group slightly below 5% reduction. However, with no dose dependency this fact was considered to have no toxicological significance and differences arise from individual variability. Individual body weight loss and average body weight loss of the rest of the groups were within 5%.
Ear thickness of the animals was measured using a thickness gauge on Days 1, 3 and 6. Increased ear thickness values (>25%) were detected on Day 6 in the 100 and 50% (w/v) groups. No increased ear thickness values (>25%) were detected in any of the animals at the 25 and 10% (w/v) treatment groups. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals. The revealing ear punch weights in the 100% (w/v) group were outside of the historical control range, but not indicated a positive response. The rest of the ear punch weights were within the historical control range.
The draining auricular lymph nodes of the animals were visually examined: they were considered larger than normal in the 100% (w/v) group, and normal for all animals at the 50, 25, and 10% (w/v) treatment groups (subjective judgement by analogy with observations of former experiments).
Based on these observations, 25 % (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 7.3) 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)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
0.7
Test group / Remarks:
25% (w/v) test item
Key result
Parameter:
SI
Value:
1.1
Test group / Remarks:
10% (w/v) test item
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
5% (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 25, 10 and 5 % (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. Minimal amount of test item precipitate was observed on the ears of the 25 % (w/v) dose group animals from Day 2 up to Day 3 (4 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, slightly larger body weight loss (5.6 %) for 1 out of 4 animals was observed in the negative control group. This finding 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 BAL0001024 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# (%)

5611

1

Negative (vehicle) control in DMF

23.0

23.9

3.9

5620

2

21.5

20.3

-5.6

5615

3

21.2

21.8

2.8

5621

4

20.4

21.6

5.9

 

 

Mean

21.5

21.9

1.8

5613

5

BAL0001024

25% (w/v)

in DMF

22.6

21.9

-3.1

5625

6

21.6

21.3

-1.4

5617

7

21.2

20.8

-1.9

5616

8

20.8

21.0

1.0

 

 

Mean

21.6

21.3

-1.4

5624

9

BAL0001024

10% (w/v)

in DMF

22.9

23.0

0.4

5612

10

21.8

21.8

0.0

5623

11

21.4

20.4

-4.7

5626

12

20.6

20.7

0.5

 

 

Mean

21.7

21.5

-0.9

5618

13

BAL0001024

5% (w/v)

in DMF

22.6

23.5

4.0

5619

14

21.3

22.7

6.6

5622

15

21.9

21.2

-3.2

5614

16

20.5

21.9

6.8

 

 

Mean

21.6

22.3

3.5

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)

3486

3453.5

8

431.7

1.0

BAL0001024

25% (w/v)

in DMF

2549

2516.5

8

314.6

0.7

BAL0001024

10% (w/v)

in DMF

4000

3967.5

8

495.9

1.1

BAL0001024

5% (w/v)

in DMF

3636

3603.5

8

450.4

1.0

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

BAL0001024

25% (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

BAL0001024

10% (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

BAL0001024

5% (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# (%)

5475

1

100% (w/v)

19.7

19.1

-3.0

5474

2

100% (w/v)

19.5

19.4

-0.5

 

 

Mean

19.6

19.3

-1.8

5477

3

50% (w/v)

19.6

17.9

-8.7

5476

4

50% (w/v)

20.0

19.6

-2.0

 

 

Mean

19.8

18.8

-5.3

5550

1

25% (w/v)

20.0

20.4

2.0

5553

2

25% (w/v)

19.7

18.9

-4.1

 

 

Mean

19.9

19.7

-1.0

5552

3

10% (w/v)

19.7

19.5

-1.0

5551

4

10% (w/v)

19.7

19.3

-2.0

 

 

Mean

19.7

19.4

-1.5

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

5475

1

100% (w/v)

0.20

0.20

NA

0.27

0.44

0.46

23.65

5474

2

100% (w/v)

0.20

0.20

0.24

0.24

0.40

0.44

25.76

5477

3

50% (w/v)

0.20

0.20

0.23

0.22

0.40

0.39

19.77

5476

4

50% (w/v)

0.20

0.20

0.22

0.23

0.33

0.28

17.21

5550

1

25% (w/v)

0.23

0.22

0.23

0.22

0.22

0.22

15.01

5553

2

25% (w/v)

0.23

0.22

0.24

0.25

0.22

0.24

14.84

5552

3

10% (w/v)

0.22

0.22

0.24

0.23

0.21

0.22

14.77

5551

4

10% (w/v)

0.21

0.22

0.22

0.23

0.23

0.22

17.87

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

100%

(w/v)

1

5475

Before treatment: symptom-free, ES: 0

After treatment: symptom-free*, ES: 0

2

5474

Before treatment: symptom-free, ES: 0

After treatment: symptom-free*, ES: 0

50%

(w/v)

3

5477

Before treatment: symptom-free, ES: 0

After treatment: symptom-free*, ES: 0

4

5476

Before treatment: symptom-free, ES: 0

After treatment: symptom-free*, ES: 0

25% (w/v)

1

5550

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

2

5553

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

10% (w/v)

3

5552

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

4

5551

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

DAY 2

100%

(w/v)

1

5475

Before treatment: symptom-free**, ES: 0

After treatment: symptom-free*, ES: 0

2

5474

Before treatment: symptom-free**, ES: 0

After treatment: symptom-free*, ES: 0

50%

(w/v)

3

5477

Before treatment: symptom-free, ES: 0

After treatment: symptom-free*, ES: 0

4

5476

Before treatment: symptom-free, ES: 0

After treatment: symptom-free*, ES: 0

25% (w/v)

1

5550

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

2

5553

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

10% (w/v)

3

5552

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

4

5551

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

DAY 3

100%

(w/v)

1

5475

Before treatment: symptom-free*, ES: 0

After treatment: symptom-free*, ES: 0

2

5474

Before treatment: symptom-free*, ES: 0

After treatment: symptom-free*, ES: 0

50%

(w/v)

3

5477

Before treatment: symptom-free, ES: 0

After treatment: symptom-free*, ES: 0

4

5476

Before treatment: symptom-free, ES: 0

After treatment: symptom-free*, ES: 0

25% (w/v)

1

5550

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

2

5553

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

10% (w/v)

3

5552

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

4

5551

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

DAY 4

100%

(w/v)

1

5475

Symptom-free***, ES: 0

2

5474

Symptom-free***, ES: 0

50%

(w/v)

3

5477

Symptom-free, ES: 0

4

5476

Symptom-free, ES: 0

25% (w/v)

1

5550

Symptom-free, ES: 0

2

5553

Symptom-free, ES: 0

10% (w/v)

3

5552

Symptom-free, ES: 0

4

5551

Symptom-free, ES: 0

DAY 5

100%

(w/v)

1

5475

Alopecia around the ears**, ES: 0

2

5474

Alopecia around the ears**, ES: 0

50%

(w/v)

3

5477

Alopecia around the ears, ES: 0

4

5476

Symptom-free, ES: 0

25% (w/v)

1

5550

Symptom-free, ES: 0

2

5553

Symptom-free, ES: 0

10% (w/v)

3

5552

Symptom-free, ES: 0

4

5551

Symptom-free, ES: 0

DAY 6

100%

(w/v)

1

5475

Alopecia around the ears, ES: 0

2

5474

Alopecia around the ears, ES: 0

50%

(w/v)

3

5477

Alopecia around the ears, ES: 0

4

5476

Alopecia around the ears, ES: 0

25% (w/v)

1

5550

Symptom-free, ES: 0

2

5553

Symptom-free, ES: 0

10% (w/v)

3

5552

Symptom-free, ES: 0

4

5551

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, BAL0001024, 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 aim of this study was to determine the skin sensitisation potential of BAL0001024 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.

 

The solubility of the test item was examined in a short Preliminary Compatibility Test. The following standard OECD vehicles were assessed: Acetone: Olive oil 4:1 (v/v) mixture, N,N-dimethylformamide (abbreviated as DMF). The best vehicle taking into account the test item characteristics, its usage and the requirements of the relevant OECD guideline was considered to be DMF. The 100% (w/v, i.e. 1 g per mL with added vehicle) dilution was the highest concentration suitable for the test. The 100% (w/v) formulation appeared, by visual examination, to be a solution.

 

A Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using four doses (2 animals/dose): 100, 50, 25 and 10 % (w/v) in DMF and based on the results, 25 % (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 BAL0001024 (formulated in DMF) at either 25, 10or 5 % (w/v),

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

-a positive control group received 25 % (w/v) HCA (dissolved in DMF). (To minimize 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. A minimal amount of test item precipitate was observed on the ears of the 25 % (w/v) dose group animals from Day 2 up to Day 3 (4 out of the 4 animals).

 

No marked group mean body weight losses (≥5%) were observed in any groups.

 

The SI values were 0.7, 1.1 and 1.0 at concentrations of 25, 10 and 5 % (w/v), respectively.

The SI value for the positive control substancea-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was 7.3 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, BAL0001024, 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.