Registration Dossier

Administrative data

Description of key information

In-Vivo: LLNA: Under the conditions of the present assay, CAS 577978-76-8, tested in a suitable vehicle (DMF), was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay.

In-Vitro: KeratinoSens Test: Under the experimental conditions of this study, the test item CAS 577978-76-8 was positive in the KeratinoSens assay and therefore was considered to activate the Nrf2 transcription factor.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 November 2019 to 27 November 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
In-Vivo study carried out as substance is intended for global registration where In-Vivo data is required.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
No further details specified in the study report
Species:
mouse
Strain:
other: CBA/CaCrl
Sex:
female
Details on test animals and environmental conditions:
Species and strain: CBA/CaCrl mice
Source: Charles River UK Limited, Manston Road, Margate, CT9 4LT Kent, GB.
Hygienic level: SPF at arrival; standard housing conditions during the study
Justification of strain: On the basis of OECD No. 429 guideline, mice of CBA/Ca or CBA/J strain can be used. Females were 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: Young adults, 8 weeks old
(age-matched, within one week)
Body weight range at starting: 18.2 – 20.9 grams (the weight variation in animals in the study did not exceed ± 20 % of the mean weight)
Acclimatisation time: At least 13 days
Note: In the Preliminary Experiment mice of 10 weeks of age (21.4 – 22.1 g) were used.

Husbandry
Animal health: Only healthy animals were used for the study. Health status was certified by the veterinarian.
Housing / Enrichment: Group caging / Mice will be provided with glass tunnel-tubes
Cage type: Type II. polypropylene/ polycarbonate
Bedding / Nesting: Bedding and certified nest building material will be available to animals during the study
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.7-25.6°C
Relative humidity: 25-73%
Ventilation: 15-20 air exchange/hour
The temperature and relative humidity were continuously monitored and 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” produced by ssniff Spezialdiäten GmbH (D-59494 Soest, Germany), and Geldiet Transport (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-8200 Veszprém, József Attila u. 36., Hungary). Copies of the relevant Certificates of Analysis are retained in the Archive at Charles River Laboratories Hungary Kft.

Bedding and nesting
Bedding of certified wood chips especially designed to keep animals in the best natural environment was provided for animals during the study. SAFE 3/4 S certified wooden chips 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 (SAFE 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 Charles River Laboratories Hungary Kft.’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.
Vehicle:
dimethylformamide
Concentration:
A Preliminary Irritation/Toxicity Test was performed in CBA/CaCrl mice using two dose levels (2 animals/dose): 50% and 25% (w/v) in DMF and based on the results, 50% (w/v) dose was selected as top dose for the main test.

In the main assay, twenty-four female CBA/CaCrl mice were allocated to six groups, each group comprised four animals:
- four groups of animals received CAS 577978-76-8 at 50%, 25%, 10% and 5% (w/v, formulated in DMF) concentrations,
No. of animals per dose:
Preliminary toxicity test: 2 animals /dose
Main assay: 4 animals / dose
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/CaCrl 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.
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.

During the Preliminary Irritation / Toxicity Test no mortality occurred. In the 50% (w/v) group very slight or well-defined erythema and extensive alopecia were observed between Day 2 (after treatment) and Day 6. In the 25 (w/v) group very slight erythema was observed on Day 3 (after treatment). Test item residue or a minimal amount of test item residue was observed on the ears of the 50 and 25% (w/v) groups.

Marked body weight loss (>5% reduction of body weight) was observed only in one animal in the 50% (w/v) dose group in the preliminary test and the mean body weight loss was -5.1%. There was no marked body weight loss in the 25% (w/v) group

Ear thickness of the animals was measured using a thickness gauge on Days 1, 3 and 6. No increased ear thickness value (>25%) was detected. Ear punch weights of the animals of both dose groups, were within the acceptable range.

The draining auricular lymph nodes of the animals were visually examined: they were larger than normal in both dose groups (subjective judgement by analogy with observations of former experiments).

Despite the marked body weight loss in one mouse in the 50% (w/v) group, this concentration was not excluded from the experiment since earlier experience shows that it can appear in a control group as well. Excluding this group from the main test could lead to a false negative result.
Based on these results, 50% (w/v) dose was selected as top dose for the main test and four concentrations was considered appropriate, in order to avoid further animal testing in case the 50% (w/v) proves to be irritant in the main test.

No ear thickness measurements or ear punch weight determination was performed in 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.

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.

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 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 4oC), 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.

EVALUATION OF THE RESULTS
DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value ("DPM"). The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as background DPM value. The results were expressed as "DPN" (DPM divided by the number of lymph nodes) following the industry standard for data presentation.
Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.
Since the test item gave a positive response and data permitted, the EC3 value of the test item was calculated (EC3 means the effective chemical concentration required for SI=3). The calculation of the EC3 value was conducted by log-linear interpolation.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The result of the positive control substance α-Hexylcinnamaldehyde (HCA) was used to demonstrate the appropriate performance of the assay. The positive control substance was examined at a concentration of 25% (w/v) in the relevant vehicle (DMF) using CBA/CaCrl 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 6.9) 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 within the historical control range. Each test item treated and control group included 4 animals.
Key result
Parameter:
SI
Value:
14.6
Test group / Remarks:
50% (w/v)
Key result
Parameter:
SI
Value:
8
Test group / Remarks:
25% (w/v)
Key result
Parameter:
SI
Value:
7.3
Test group / Remarks:
10% (w/v)
Key result
Parameter:
SI
Value:
5.3
Test group / Remarks:
5% (w/v)
Cellular proliferation data / Observations:
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.

CLINICAL OBSERVATION

There was no mortality observed during the main assay. In the 50% (w/v) group very slight erythema and extensive alopecia were observed between Day 3 (before treatment) and Day 6. No clinical signs were observed in any other group. Minimal amount of test item residue was observed on the ears of the 50% (w/v) groups on Day 3 after treatment. No test item residue was observed in any other group.

BODY WEIGHT MEASUREMENT

No test item related effect was noted on body weight.

PROLIFERATION ASSAY

The appearance of the lymph nodes was larger than normal in the 50% (w/v) group and positive control group, slightly larger than normal in the 25% (w/v) group and normal in the negative control group and in the other test item treated dose groups.

The stimulation index values were 14.6, 8.0, 7.3 and 5.3 at concentrations of 50, 25, 10 and 5% (w/v), respectively.

INTERPRETATION OF OBSERVATIONS

The test item was solid, which was applied 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 were considered to be good evidence that CAS 577978-76-8 is a sensitizer.

The dose response does not allow a reliable extrapolation to the EC3 value, although it appears to be above 2% (calculated EC3 equals to 2.3% suggesting a Cat.1B). To be sure of the classification a further group at 2% test item would be required.

Based on the observed results, the test item needs classification of Category 1 according to the GHS or CLP

Summary of Preliminary Study Data

Concentrations

Physical Formulation

Clinical Observations

Body Weight

Erythema

Ear Thickness

Ear Biopsy weight

50% (w/v)

A

A

E

A

A

A

25% (w/v)

A

A

A

A

A

A

Notes: U=Unacceptable; A=Acceptable; E=Equivocal; NM=Not measured

Experimental Groups and Treatments

Groups

Test item concentration

(% w/v)

No. of animals

Negative (vehicle) control (DMF)

---

4

50% (w/v) in DMF

50

4

25% (w/v) in DMF

25

4

10% (w/v) in DMF

10

4

5% (w/v) in DMF

5

4

Positive (25% (w/v) HCA in DMF)

---

4

Individual Body Weights for all Animals with Group Means

Identity Number

Animal Number

Test Group Name

Initial Body Weight (g)

Terminal Body Weight* (g)

Change# (%)

1

8880

 

Negative (vehicle) control (DMF)

20.4

21.3

4.4

2

8891

20.4

21.3

4.4

3

8890

19.0

19.9

4.7

4

8879

18.3

18.9

3.3

 

 

Mean

19.5

20.4

4.2

5

8881

 

CAS 577978-76-8

50% (w/v) in DMF

20.4

21.2

3.9

6

8899

19.9

20.5

3.0

7

8889

19.3

19.4

0.5

8

8878

19.7

21.0

6.6

 

 

Mean

19.8

20.5

3.5

9

8892

 

CAS 577978-76-8

25% (w/v) in DMF

20.5

21.0

2.4

10

8893

19.9

20.6

3.5

11

8877

19.5

21.2

8.7

12

8888

18.5

18.7

1.1

 

 

Mean

19.6

20.4

4.0

13

8900

 

CAS 577978-76-8

10% (w/v) in DMF

20.9

22.0

5.3

14

8894

19.3

18.7

-3.1

15

8883

19.1

19.3

1.0

16

8887

18.3

19.0

3.8

 

 

Mean

19.4

19.8

1.8

17

8895

 

CAS 577978-76-8

5% (w/v) in DMF

20.4

20.8

2.0

18

8882

19.3

19.8

2.6

19

8886

19.3

19.6

1.6

20

8896

18.2

19.0

4.4

 

 

Mean

19.3

19.8

2.6

21

8885

 

CAS 577978-76-8

25% (w/v) HCA in DMF

20.1

20.3

1.0

22

8898

20.0

22.0

10.0

23

8897

19.3

20.7

7.3

24

8884

18.2

18.9

3.8

 

 

Mean

19.4

20.5

5.5

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

No. of Nodes

DPN

Stimulation Index Values

Background (5 (w/v) % TCA)

34

---

---

---

---

Negative control (DMF)

4097

4063.0

8

507.9

1.0

50% (w/v)

59206

59172.0

8

7396.5

14.6

25% (w/v)

32657

32623.0

8

4077.9

8.0

10% (w/v)

29564

29530.0

8

3691.3

7.3

5% (w/v)

21529

21495.0

8

2686.9

5.3

Positive control (25% HCA)

28158

28124.0

8

3515.5

6.9

 The stimulation index values were 14.6, 8.0, 7.3 and 5.3 at concentrations of 50, 25, 10 and 5% (w/v), respectively.

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Under the conditions of the present assay, CAS 577978-76-8, tested in a suitable vehicle (DMF), was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay.
Executive summary:

The object of this study was to determine the skin sensitisation potential of CAS 577978-76-8 following dermal exposure in mice. The study was being performed with vertebrate animals because the chemical nature of the test item is not compatible with the available in vitro alternative tests*. The in vitro testing was considered not to be technically feasible.

*IMPORTANT NOTE: With Commission Regulation (EU) 2016/1688 adopted on the 20th September 2016, the testing strategy for the assessment of the skin sensitisation 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 best vehicle taking into account the test item characteristics and the requirements of the relevant OECD guideline was considered to be N,N-dimethylformamide (DMF). The 50% formulation was the highest concentration which was suitable for the preliminary test.

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

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

Four groups of animals received CAS 577978-76-8 at 50%, 25%, 10% and 5% (w/v, formulated in DMF) concentrations,

A negative control group received the vehicle (DMF) only,

A positive control group received 25% (w/v) HCA (formulated in DMF).

The formulations 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 animals were 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 observed during the main assay. In the 50% (w/v) group very slight erythema and extensive alopecia were observed between Day 3 (before treatment) and Day 6. No clinical signs were observed in any other group. Minimal amount of test item residue was observed on the ears of the 50% (w/v) groups on Day 3 after treatment. No test item residue was observed in any other group.

No test item related effect was noted on body weight.

The SI values were 14.6, 8.0, 7.3 and 5.3 at concentrations of 50, 25, 10 and 5% (w/v), respectively.

The dose response does not allow a clear classification as 1A or 1B, only as Cat.1.

The DPN values of the negative control group was in line with historical control data. The SI value for the positive control substance α-Hexylcinnamaldehyde (HCA), formulated in the same vehicle as the test item (SI=6.9) demonstrated the appropriate performance of the assay, therefore confirmed the validity of the assay.

In conclusion, under the conditions of the present assay, CAS 577978-76-8, tested in a suitable vehicle (DMF), was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay.

The study result triggers the following classification/labelling:

Regulation (EC) No 1272/2008 (CLP): Category 1

GHS (rev. 8) 2019: Category 1

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 July 2019 to 23 August 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
With Commission Regulation (EU) 2016/1688 adopted on the 20th September 2016, the testing strategy for the assessment of the skin sensitisation 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.
Specific details on test material used for the study:
No further details specified in the study report
Details on study design:
Vehicle and negative control
Based on the results of the solubility assay, the selected vehicle was Dimethylsulfoxide (DMSO).
This vehicle was used as the negative control, and was applied to cells at a concentration of 1% in culture medium. Moreover and since several test items were assayed concurrently, the results of this negative control were shared.

Positive control
Name: Cinnamic Aldehyde (CA)
Synonym: trans-Cinnamaldehyde
CAS number: 14371-10-9
Storage conditions: at +4°Cand under nitrogen gas.
Since several test items were assayed concurrently, the results of this positive control were shared.
For each run, the positive control item was dissolved in DMSO to a final concentration of 200 mM. This solution was further diluted to a final concentration of 6.4 mM. Then, it was diluted in DMSO by serial dilutions in the Master plate 100x, using a dilution factor of 2, to obtain a total of 5 concentrations. Subsequently, each formulation of the Master plate 100x was diluted 25-fold in treatment medium in another 96-well plate called "Master plate 4x". The final tested concentrations ranged from 4 to 64 µM. All these formulations were prepared within 4 hours before use, then kept at room temperature and protected from light until use.
est item formulations
The test item was first ground into a fine powder using a pestle and a mortar; and was then dissolved in DMSO at the selected concentration in the run (i.e. 40 mg/mL or 1.5 mg/mL).
One stock formulation was prepared for each run. It was then diluted in DMSO by serial dilutions, using a dilution factor of 2 or 1.41, to obtain a total of 12 concentrations in a 96-well plate; this 96-well plate was called "Master plate 100x". Subsequently, each formulation of the Master plate 100x was 25-fold diluted in treatment medium in another 96-well plate called "Master plate 4x" taking care to adjust all wells to the same DMSO level.
All formulations were prepared within 4 hours before use, and kept at room temperature and protected from light until use.

TEST SYSTEM
KeratinoSens cells: the cell line KeratinoSens is stably transfected with a modified plasmid. This plasmid contains an ARE sequence from the AKR1C2 gene and a SV40 promotor which are inserted upstream of a luciferase gene. The resulting plasmid was transfected into HaCaT keratinocytes and clones with a stable insertion selected in the presence of Geneticin / G-418. Induction of the luciferase gene is the endpoint evaluated and reflects the activation by the test item of the Nrf2 transcription factor in this test.
Supplier: cell line provided by Givaudan.
Batch: D1.
Storage condition: at -80°C.
Mycoplasm: absence of mycoplasm was confirmed.

STUDY DESIGN
The test item was tested in two independent validated runs using cells of a different passage number. The plates were processed as described below:

Solubility assay
A solubility assay was performed prior to the first treatment to select the most appropriate vehicle (between DMSO, water for injections or treatment culture medium).
A 10-minute sonication step was used to help solubilize the test item in some vehicles.
Once a solution or a stable dispersion was obtained in the vehicle(s), the formulation was 100-fold diluted in culture medium. Then, a visual inspection of the samples was performed immediately as well as after an overnight period of incubation at 37°C to evaluate the presence or absence of precipitate/emulsion.

Method for a run of the KeratinoSens assay
Cell seeding for testing
Cells were grown using general culture procedures up to 80-90% confluence,
the day prior to treatment, cells were washed twice with D-PBS containing 0.05% EDTA, harvested, re-suspended in Maintenance medium No. 2 and counted using Trypan Blue dye. Cell concentration was adjusted to a density of 8 x 104 cells/mL,
cells were then distributed into four 96-well plates (three white plates and one transparent plate), by adding 125 µL (representing 1 x 104 cells) per well taking care to avoid sedimentation of the cells during seeding,
after seeding, the cells were grown for 24 hours (± 1 hour) in 96-well microtiter plates prior to test item addition.

Treatment
After the 24-hour growing period, the medium was removed by aspiration and replaced by 150 µL of treatment medium,
from the Master plate 4x. 50 µL was added to each well of the three white assay plates and 50 µL to the transparent plate for cytotoxicity evaluation, all plates were covered by a sealing membrane to avoid any evaporation of volatile test items and also to avoid any cross-contamination between wells.
The plates were then incubated for 48 hours (± 2 hours) at 37°C, 5% CO2, 90% humidity.

Endpoint measurements
Microscopic observations to evaluate the presence or absence of precipitate:
In the transparent plate After 48 hours (± 2 hours) incubation period, the presence or absence of precipitate/emulsion was determined in each well by microscopic inspection.

Luminescence flash signal to evaluate induction signal - white plates
After incubation, the supernatants from the white assay plates were discarded, the cells were washed once with D-PBS, a volume of 20 µL of passive lysis buffer was added to each well and the cells were incubated for 20 minutes (± 2 minutes) at room temperature under orbital shaking, the plates containing the passive lysis buffer were then placed in the luminometer for reading using the following program:
50 µL of the luciferase substrate was added to each well,
1 second after this addition, the luciferase signal was integrated for 2 seconds.

Absorbance signal to evaluate cytotoxicity - transparent plate
For the cell viability assay plate (transparent plate), the medium was removed by aspiration and was replaced by 200 µL of treatment medium, 27 µL of MTT solution at 5 mg/mL in D-PBS was then added to each well, the plates were covered with a sealing membrane and returned at 37°C in the incubator in a humidified atmosphere for 4 hours (± 10 minutes), at the end of the incubation period, the medium was removed and 200 µL of a 10% SDS solution was added to each well, the plates were covered with a sealing membrane and placed at 37°C in the incubator in a humidified atmosphere for an overnight period to extract the formazan from cells, after the overnight incubation, the absorption of each well was determined at 600 nm using the plate reader.
Positive control results:
Concentration: 4, 8, 16, 32, 64 (µM)
Geometric mean: 103, 111, 117, 121, 116
Key result
Parameter:
other: IC30
Remarks:
geometric means of the two runs
Run / experiment:
Cytotoxicity
Value:
3.69
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: IC50
Remarks:
geometric means of the two runs
Run / experiment:
Cytotoxicity
Value:
4.81
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
Data evaluation was performed using a validated Excel sheet. The generated raw data (luminescence data for the luciferase activity and absorbance data for the MTT test) were pasted into an Excel template, and all data processing performed automatically.
For the MTT and the luciferase data, the background value recorded in the empty well without cells (blank) was subtracted.
For the MTT data, the % viability was calculated for each well in the test plate in relation to average of the six negative control wells.
For the luciferase data, the average value of the six negative control wells was set to 1 and for each well in the plate, the fold induction was calculated in relation to this value.
For wells in which a statistically significant gene-induction (using a student test, also called T-test) over the 1.5 threshold was found, the following parameters were calculated from the processed raw data: Imax: Maximal induction factor of luciferase activity compared to the negative control over the complete dose-response range measured.
EC1.5: Concentration at which a 1.5-fold luciferase gene induction is obtained,
IC50 and IC30: Concentrations effecting a reduction of cellular viability by 50% and 30%, Indication whether a significant 1.5-fold gene induction occurred below the IC30.
The data were plotted in graphs and the Imax and the EC1.5 values were visually checked since uneven dose-response curves or large variation may lead to incorrect extrapolation.
Also, the individual and overall geometric means (IC50 and IC30) were calculated, when applicable.

All acceptance criteria were fulfilled in each run, both runs were therefore considered as validated.

The evaluation criteria for a positive response are met in both runs, the final outcome is therefore positive. This positive result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment. It cannot be used on its own to conclude on a skin sensitization potential.

SOLUBILITY ASSAY

In the solubility assay, the test item was found soluble in DMSO at both tested concentrations (i.e. 20 mg/mL and 40 mg/mL) and the resulting formulations were reported as orange solutions.  

In contrast, heterogeneous suspensions were obtained when tested at 20 mg/mL and 40 mg/mL in water for injections or in culture medium, despite a 10-minute sonication step.

Since solutions were obtained at 20 mg/mL and 40 mg/mL in DMSO, these stock formulations were diluted in the treatment culture medium (i.e final concentrations of 200 µg/mL and 400 µg/ml, respectively). A slight to strong precipitate was observed immediately and was still noted after the overnight period of incubation at 37°C.  

Based on these results, the test item stock formulation to be used in the first run was therefore selected to be 40 mg/mL in DMSO.

First run

The first run was performed using the following concentrations: 0.20, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, 200 and 400 µg/mL in culture medium containing 1% DMSO.

At these tested concentrations:

Moderate to strong precipitate was observed in treated wells at concentrations ≥ 50 µg/mL at the end of the 48-hour treatment period,

Cytotoxicity (i.e. cell viability < 70%) was noted at concentrations ≥ 6.25 µg/mL. The corresponding IC30 and IC50 were calculated at 3.53 and 4.75 µg/mL, respectively,  

Statistically significant gene-fold inductions above the threshold of 1.5 were noted in comparison to the negative control at concentrations ≥ 1.56 µg/mL, and up to the cytotoxic concentrations, with an overall dose response relationship. The corresponding Imax and EC1.5 were calculated at 61.06 µg/mL and 1.04 µg/mL, respectively.

This first run is therefore considered as positive.

Second run

Due to the cytotoxicity observed in the first run, a lower and narrower range of concentrations was used in the second run, and the following concentrations were tested: 0.34, 0.48, 0.68, 0.96, 1.35, 1.91, 2.69, 3.80, 5.35, 7.54, 10.6 and 15 µg/mL in culture medium containing 1% DMSO.

At these tested concentrations:

no precipitate/emulsion was observed in any test item-treated wells at the end of the 48-h treatment period,

cytotoxicity (i.e. cell viability < 70%) was noted at concentrations ≥ 5.35 µg/mL. The corresponding IC30 and IC50 were calculated at 3.87 and 4.87 µg/mL, respectively,  

statistically significant gene-fold inductions above the threshold of 1.5 were noted in comparison to the negative control at concentrations of 1.91 µg/mL and from 3.80 µg/mL to the cytotoxic concentrations, with an overall dose response relationship. The corresponding Imax and EC1.5 were calculated at 59.40 µg/mL and 1.48 µg/mL, respectively.

This second run is therefore also considered as positive.

The geometric means IC30 and IC50 of the two validated runs were calculated to be 3.69 and 4.81 µg/mL, respectively.

Imax, IC30, IC50 and EC1.5 values (mean and SD values) obtained after treatment with the test item in both runs 

CAS 577978-76-8

Imax

EC1.5

(μg/mL)

IC50

(μg/mL)

IC30

(μg/mL)

Run 1

61.06

1.04

4.75

3.53

Run 2

59.40

1.48

4.87

3.87

Mean

60.23

n.r.

n.r.

n.r.

Geometric Mean

n.r.

1.24

4.81

3.69

SD

1.18

0.31

0.09

0.24

n.r.: not requested by the OECD Guideline

Evaluation of the viability (%) of cultures treated with the positive control in both runs

cinnamic aldehyde

 

Concentrations (μM)

4

8

16

32

64

Viability (%) in Run 1

101

105

114

120

123

Viability (%) in Run 2

105

117

119

122

109

Mean viability (%)

103

111

117

121

116

Geometric Mean (%)

103

111

117

121

116

SD

3

9

3

2

10

Gene induction values, Imax, IC30, IC50 and EC1.5 values obtained with the positive control in both runs

cinnamic aldehyde

Concentrations (μM)

Imax

EC1.5

(μM)

IC50

(μM)

IC30

(μM)

4

8

16

32

64

Viability (%) in Run 1

1.7

1.8

1.9

3.1

4.3

4.28

3.43

-

-

Viability (%) in Run 2

1.4

1.4

2.1

3.3

6.7

6.71

9.38

-

-

Mean viability (%)

1.5

1.6

2.0

3.2

5.5

5.50

n.r.

n.r.

n.r.

Geometric Mean (%)

n.r.

n.r.

n.r.

n.r.

n.r.

n.r.

5.67

-

-

SD

0.2

0.3

0.1

0.1

1.7

1.72

4.21

-

-

 

-.: no data available

n.r.: not requested by the OECD Guideline

Luminescence values for the negative control wells and the %CV between these values in both runs 

Negative control

Luminescence reading

Mean

%CV

 

Run 1

Replicate 1

252337

323629

305527

326757

272337

303292

 

284207

 

11.12

Replicate 2

289478

267381

328639

342733

257759

287498

Replicate 3

274059

261756

272673

270040

226601

253223

 

Run 2

Replicate 1

619727

372387

501603

598433

559846

525192

 

450673

 

17.94

Replicate 2

383970

369407

376592

470281

414381

430644

Replicate 3

439281

410463

478052

416408

368489

376966

 

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Under the experimental conditions of this study, the test item CAS 577978-76-8 was positive in the KeratinoSens assay and therefore was considered to activate the Nrf2 transcription factor.
Executive summary:

The objective of this study was to evaluate the potential of the test item, CAS 577978-76-8, to activate the Nrf2 transcription factor. This test is a part of a tiered strategy for the evaluation of skin sensitisation potential. The design of this study was based on OECD Test Guideline No. 442D. The study was performed in compliance with Citoxlab France standard operating procedures and with the OECD Principles of Good Laboratory Practice.

Principle

This in vitro test uses the KeratinoSens cell line, an immortalized and genetically modified Human adherent HaCaT keratinocyte cell line. The KeratinoSens cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence. Sensitizers with electrophilic properties provoke the dissociation of Keap-1 from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase.

Methods

The KeratinoSens cells were first plated on 96-well plates and grown for 24 hours at 37°C. Then the medium was removed and the cells exposed to the vehicle control or to different concentrations of test item and positive controls. The treated plates were then incubated for 48 hours at 37°C. At the end of the treatment, cells were washed and the luciferase production measured by flash luminescence. In parallel, cytotoxicity was measured by a MTT reduction, this was taken into consideration in the interpretation of the sensitisation results. Two independent validated runs were performed as part of this study.

Results

All acceptance criteria were met for both runs; the study was therefore considered as validated.

This study was performed at concentrations from 0.20 to 400 µg/mL in culture medium containing 1% DMSO.

At these tested concentrations, statistically significant gene-fold inductions above the threshold of 1.5 were noted in comparison to the negative control at concentrations ≥ 1.56 µg/mL with an apparent dose-response relationship, in both runs.  

Since the evaluation criteria for a positive response are met in both runs, the final outcome is therefore positive. This positive result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment. It cannot be used on its own to conclude on a skin sensitization potential.

Conclusion

Under the experimental conditions of this study, the test item CAS 577978-76-8 was positive in the KeratinoSens assay and therefore was considered to activate the Nrf2 transcription factor.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The study result triggers the following classification/labelling:

Regulation (EC) No 1272/2008 (CLP): Category 1

GHS (rev. 8) 2019: Category 1