Diallyl hexahydrophthalate

Endpoint summary

Description of key information

Due to the adverse effects observed the in one of the vitro studys (OECD 442E) an in vivo study was conducted. The LLNA confirmed that the there is no sensitisation potential, i.e a non sensitizer

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 December 2017 to 19 January 2018 (Experimental start to experimental completion)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD guideline No. 442E: "In vitro skin sensitization: human Cell Line Activation Test (h-CLAT)", 29 July 2016.

Deviations:

yes

Remarks:

Main test: Run B, treated plates incubated for 24h 57 min, not 24h ±30 min. Acceptance criteria: Run B (18.1.18), positive control cells did not show RFI-CD86 & RFI-CD54 above threshold. Cells from (Run A, 25.1.18) responded. No impact to study..

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Human Cell Line Activation Test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Osaka Soda Co., Ltd
- Batch No.of test material: 40201
- Expiration date of the batch: 26 January 2019
- Purity test date: 7 October 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protcected from light
- Stability under test conditions:assumed stable for the duration of the study
- Solubility and stability of the test substance in the solvent/vehicle: soluble in DMSO (upto 500 mg/mL), stability of the test item in DMSO not determined.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

The study was divided in two successive phases.
1), a Dose-Range Finding assay (DRF) was performed to assess test item toxicity and, etermine the CV75 ( test item concentration resulting in 75% cell viability compared to the vehicle control).
2) based on cytotoxicity data obtained from the DRF, a concentration series was tested in successive runs in the main test.
At each phase, all information relating to test item concentrations and run identification were given by the Study Director in the study files and no study plan amendment was issued for that purpose.

Dose-Range Finding assay (DRF)

The DRF consisted in two separated assays.
Treatments of DRF assays were performed at the following concentrations (final concentrations): 7.81, 15.63, 31.25, 62.50, 125, 250, 500 and 1000 μg/mL.
Each assay was performed as described here below.
Test item stock solutions were prepared at 8 different concentrations by 2-fold dilutions using the selected vehicle (DMSO). These stock formulations were then diluted 250-fold into cRPMI to obtain working solutions.
The working solutions were finally used for exposure by adding 500 μL of working solutions to the volume of THP-1 cell suspension in the plate (500 μL) to achieve a further 2-fold dilution. In order to avoid evaporation of volatile chemicals and cross-contamination between wells, a sealer was placed on each 24-well plate just after treatment, before putting the plastic lids back on each plate.
The treated plates were incubated for 24 hours ± 30 minutes in a humidified incubator set at 37°C and 5% CO2.
At the end of the treatment phase, cells were transferred into sample tubes and collected by centrifugation. The supernatants were discarded and the remaining cells were resuspended with 600 μL of FACS buffer. Finally, cells were resuspended in 200 μL FACS buffer and the plate was positioned into the plate-reader of the flow cytometer. A volume of 10 μL of Propidium Iodide (PI) solution at 12.5 μg/mL was added automatically by the flow cytometer before acquisition of a sample to obtain a final PI concentration of 0.625 μg/mL per well.

Main test

The main test consisted in two separated runs being performed as described here below.
Test item stock solutions were prepared at 8 different concentrations by 1.2-fold dilutions using the selected vehicle. The highest concentration corresponded to 1.2-fold the mean CV75. The maximum concentration in the plates was 676.33 μg/mL.
All stock formulations were then 250-fold diluted into cRPMI to obtain working solutions.
In parallel, the working solutions of positive controls DNCB and NiSO4 and vehicle control were prepared..
All working solutions were finally used for exposure by adding 500 μL of working solutions to the volume of THP-1 cell suspension in the plate (500 μL) to achieve a further 2-fold dilution. In order to avoid evaporation of volatile chemicals and cross-contamination between wells, a sealer was placed on each 24-well plate just after treatment, before putting the plastic lids back on each plate.
The treated plates were then incubated for 24 hours ± 30 minutes (see § Study plan adherence) in a humidified incubator set at 37°C and 5% CO2.
During the main test, treatments were performed at the following concentrations (final concentrations in the wells): 188.75, 226.5, 271.8, 326.16, 391.39, 469.67, 563.61 and 676.33 μg/mL.
At the end of the treatment phase, cells were transferred into sample tubes and collected by centrifugation, washed twice with 1 mL FACS buffer and blocked with 600 μL of blocking solution and incubated at 4°C for 15 minutes (± 1 minute). After blocking, cells were split in three aliquots of 180 μL into a 96-well round bottom plate and centrifuged before staining with antibodies. A volume of 50 μL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies prepared in FACS buffer was added to each aliquot before incubation for 30 minutes (± 2 minutes) at 4°C.Finally, cells were washed with 150 μL FACS buffer 2 times and re-suspended in 200 μL FACS buffer. The plate was then positioned into the plate-reader of the flow cytometer. A volume of 10 μL of PI solution at 12.5 μg/mL was added automatically by the flow cytometer before acquisition of a sample to obtain a final PI concentration of 0.625 μg/mL per well.

Positive control results:

Reference chemicals were correctly classfied in a maximum of three validated runs using both working cell banks.

Key result

Run / experiment:

other: Run A

Parameter:

other: RFI CD86

Value:

391.4

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: Run A

Parameter:

other: RFI CD86

Value:

271.8

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: Run A

Parameter:

other: RFI CD54

Value:

226.5

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: Run B

Parameter:

other: RFI CD54

Value:

676.3

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

Run B: RFI CD86 did not exceed the positivity thresholds at any tested concentration.

Interpretation of results:

study cannot be used for classification

Remarks:

Part of the tiered strategy for skin sensitization assessment

Conclusions:

Under the experimental conditions of this study, the test item, MDAC, was found to be positive in the h-CLAT test method

Executive summary:

The objective of the study was to determine the ability of the test item to induce an increase in cell surface markers expression in THP-1 cells using the h-CLAT test method.

A solubility assessment was first performed in 0.9% NaCl and DMSO to select the vehicle and highest concentration to be used for test item formulation preparations. Following the solubility assays, the cytotoxic potential was assessed in a Dose-Range Finding assay in order to select sub-toxic concentrations for testing in the main test. The skin sensitizing potential of the test item was then tested in the main test in two successive runs.

In each run, the test item formulations were applied to THP-1 cells and cultured in a 24-well plate for 24h ± 30 minutes at 37°C, 5% CO2 in a humidified incubator. A set of control wells was also added in each plate to guarantee the validity of each run. At the end of the incubation period, cells from each well were distributed to three wells of 96-well plate: the first well was labeled with IgG1-FITC antibodies, the second one was labeled with CD86-FITC antibodies and the third one was labelled with CD54-FITC antibodies. Then, just before flow cytometry analysis of CD86 and CD54 expression, all cells were dyed with Propidium Iodide for viability discrimination.

For each run, the Mean Fluoresence Intensity (MFI) obtained for each test sample was corrected by the isotype control IgG1 MFI value to obtain the corrected MFI. Corrected MFI value from the corresponding vehicle control was set to 100% CD54 and CD86 expression by default. Then, corrected MFI values from each test sample were compared to the corresponding vehicle control to obtain the Relative Fluorescence Index for CD86 and CD54 expression for each tested concentration (RFI CD86 and RFI CD54).

Results showed that the test item was found soluble in DMSO at the concentration of 500 mg/mL The test item induced a decrease in cell viability < 75% in both DRF runs and the calculated mean CV75 value was: 563.61 μg/mL. The highest concentration tested in the main test was therefore 676.33 μg/mL (i.e. 1.2-fold the mean CV75).

Under the experimental conditions of this study, the test item, MDAC, was found to be positive in the h-CLAT test method.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

09 February 2017 - 09 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Direct Peptide Reactivity Assay (DPRA)

Justification for non-LLNA method:

This test is part of a tiered strategy for skin sensitization assessment.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name: MDAC
- Description: Colorless liquid
- CAS No. 13846-31-6
- Source and batch No. of test material: Osaka Soda Co., Ltd. Batch No. 40201
- Expiration date of the batch: 26 January 2019
- Purity test date: 99.2%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protected from light
- Stability under test conditions: Assumed stable for the duration of the study
- Solubility and stability of the test substance in the solvent/vehicle: The test item was soluble (without sonication) at 100 mM in acetonitrile. Assumed stable for the duration of the study
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was pre-weighed and stored under appropriate conditions until testing. It was dissolved in the selected vehicle (acetonitrile) at 100 mM. The formulation was used just after preparation.

Details on the study design:

Skin sensitisation (In chemico test system)

The assay determines the chemical reactivity of the test item to cysteine and lysine peptides, which is a unifying characteristic of most skin sensitizing chemicals.

Details on study design: The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following 24-hour contact between the test item and synthetic cysteine and lysine peptides in acetonitrile at ratios 1:10 cysteine:test item and 1:50 lysine:test item. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with UV detection at 220 nm. Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent).

The direct peptide reactivity assay was completed in one run. Samples were prepared in triplicate (reference control, positive control and test item), except for co-elution control samples (co-elusion control with cysteine and co-elusion with lysine) for which only one sample was prepared per peptide buffer. All samples were incubated for 24 (+/-2) hours at 25ºC protected from light before injection into the HPLC/UV system. After the incubation period and before HPLC analysis, a visual inspection of samples was performed to detect precipitate or phase separation. Samples presenting preciptate and/or phase separation were centrifuged at 400g for 5 minutes at room temperature and only supernatents were injected into the HPLC/UV system. Otherwise, the vials were directly transferred onto the HPLC/UV system.

One set of calibration standards was prepared with each analytical sequence by spiking each peptide (lysine and cysteine) in seperate solutions of 20% acetonitrile:peptide dilution buffer to obtain at least six different concentration levels ranging from 0.0167 to 0.534 mM. A dilution buffer blank was also included in the standard calibration curve. Calibration curves were defined by the relationships between the peak area signal of the peptide versus the nominal concentration. These curves were obtained by using the appropriate mathematical model.

Cysteine peptide: The cysteine peptide solution was freshly prepared at 0.667 mM in an aqueous phosphate buffer (pH 7.5) solution.
. Peptide sequence : Ac-RFAACAA-COOH
. Molecular weight : 750.88 g/mol
. Supplier : JPT Peptide Technologies GmbH
. Batch No. : 111016HS_MHeW0117
. Storage condition : At -20°C
. Description : White powder

Lysine peptide: The lysine peptide solution was freshly prepared at 0.667 mM in an aqueous ammonium acetate buffer (pH 10.2) solution.
. Peptide sequence : Ac-RFAAKAA-COOH
. Molecular weight : 775.91 g/mol
. Supplier : JPT Peptide Technologies GmbH
. Batch No. : 220114HSDWW0117
. Storage condition : At -20°C
. Description : white powder

DESIGN OF THE DIRECT PEPTIDE REACTIVITY ASSAY
The test item was tested in one run. The run was processed as described below.
- Preparation of the samples: The following samples were prepared in triplicate except for the co-elution control samples for which only one sample was prepared per peptide buffer.
- Co-elution control samples preparation:
For the co-elution control with cysteine peptide: 50 μL of test item formulation was incubated with 750 μL of cysteine peptide dilution buffer (without cysteine peptide) and 200 μL of acetonitrile.
For the co-elution control with lysine peptide: 250 μL of test item formulation was incubated with 750 μL of lysine peptide dilution buffer (without lysine peptide).

Reference control samples preparation
- Reference control A and B samples. In a vial, acetonitrile was added to a volume of peptide solution (cysteine or lysine) to achieve a nominal concentration of 0.500 mM.
- Reference control C samples: Reference control C samples were prepared for each solvent used to dissolve the test and positive control items.

For the reference control C prepared with cysteine peptide: 50 μL of vehicle (acetonitrile) was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile.
For the reference control C prepared with lysine peptide: 250 μL of vehicle (acetonitrile) was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate buffer at pH 10.2).

Cinnamaldehyde (positive control) depletion control samples preparation
For the reactivity of cinnamaldehyde with cysteine peptide: 50 μL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile.
For the reactivity of cinnamaldehyde with lysine peptide: In parallel, 250 μL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).

2.4.1.4 Test item samples preparation
For the reactivity of test item with cysteine peptide: 50 μL of test item formulation was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile.
For the reactivity of test item with lysine peptide: In parallel, 250 μL of test item formulation was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).

All samples (co-elution controls, reference controls, test item and positive control samples) were then incubated during 24 (± 2) hours at 25°C and protected from light before injection into the HPLC/UV system. Study samples were assayed in batches using HPLC/UV analysis.

For each peptide, the analytical sequence included at least:
- one blank sample (peptide dilution buffer)
- one calibration curve injected at the beginning of the analytical batch
- three reference control A samples
- the co-elution control sample
- three reference control B samples
- reference control C samples (replicate 1)
- positive control sample (replicate 1)
- test item sample (replicate 1)

The injection order of the reference control C, positive control and test item study samples were reproduced identically for replicate 2 and then replicate 3:
. three reference control B samples.

Positive control results:

The positive control was Cinnamaldehyde (CAS No. 104-55-2, batch No. MKBV4784V, supplied by Sigma-Aldrich). Its molecular weight was 132.16 g/mol and the purity was 98.9%. The positive control was pre-weighed and stored under appropriate conditions until ready to perform testing. It was dissolved in acetonitrile at 100 mM and the formulation was used just after its preparation.

The determination of cysteine peptide and lysine peptide depletion in sample spiked with a solution at 100 mM of Cinnamaldehyde caused a mean depletion rate (%) of Cinnamaldehyde of 74.26%. this determines a high reactivity depletion classification.

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as study samples were satisfied. The study was therefore considered to be valid.

Key result

Run / experiment:

other: The test item was tested in one run.

Parameter:

other: Mean of Cysteine and Lysine % peptide depletion

Value:

6.38

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Since precipitate and/or micelles were observed at the end of incubation with peptides, the peptide depletion may be underestimated.

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The mean of the percent cysteine and percent lysine depletions was equal to 0.45% (which determines a no reactivity/minimal reactivity depletion classification). However, since precipitate and/or micelles were observed at the end of the incubation with the peptides, the peptide depletion may be underestimated. The DPRA prediction is considered as negative and the test item is likely not to have any potential to cause skin sensitization, though with limitations due to test item precipitation or phase separation with the peptides.

ACCEPTANCE OF RESULTS:

. the calibration curve should have a coefficient of determination (r2) ≥ 0.99,
. the mean peptide concentrations of the reference control A samples should be within ± 10% of the nominal concentration,

. the cinnamaldehyde depletion control samples should meet the following acceptance criteria:
− for the cysteine peptide, the mean percent depletion value should be between 60.8 and 100% with a SD < 14.9%,
− for the lysine peptide, the mean percent depletion value should be between 40.2 and 69.0% with a SD < 11.6%,
. the CV of the mean peptide peak area of the nine reference controls B and C samples in acetonitrile must be < 15.0%.

The test item’s results were considered valid if the following criteria were fully met:
. the mean peptide concentrations of the reference control C samples prepared in the appropriate solvent should be within ± 10% of the nominal concentration,
. the maximum SD for the test item replicates should be < 14.9% for the percent cysteine depletion value and < 11.6% for the percent lysine depletion value.

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide. For the cysteine peptide, the mean depletion value was 0.00%, for the lysine peptide, the mean depletion value was 0.90% (mean of the percent cysteine and lysine depletions was equal to 0.45%).

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Interpretation of results:

GHS criteria not met

Remarks:

Part of a tiered strategy for skin sensitization assessment.

Conclusions:

This test evaluated the reactivity of the test item (MDAC) to synthetic cysteine and lysine peptides. The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24-hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC/UV detection at 22 nm. All samples were incubated for 24 (+/-2) hours at 25ºC and protected from light before injection into the HPLC/UV system. Samples presenting preciptate and/or phase separation were centrifuged at 400g for 5 minutes at room temperature and the resulting supernatents injected into the HPLC/UV system.

Since the mean of the percent cysteine and percent lysine depletions was < 6.38%, the test item was considered to have no/minimal peptide reactivity. Therefore, the DPRA predicition is considered as negative and the test item is likely not to have any potential to cause skin sensitization, though with limitations due to test item precipitation or phase separation with the peptides.

The test item MDAC was considered to have no/minimal peptide reactivity, though with limitations due to test item precipitation or phase separation with the peptides. The test item is considered negative in the DPRA assay.

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 June to 4 September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Three in vitro assays were conducted, prior to conducting this assay (OECD 442 B, D and E). OECD 442E gave a positive responce and so futhur investigation was required to confirm if the substance required classification.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Name: MDAC
Chemical Name: 1,2-Cyclohexanedicarboxylic acid, di-2-propenyl ester
CAS No.: 13846-31-6
Batch/Lot No.: 40201
Expiry date: 26 January 2019
Purity*: 99.2%
Description: Colourless liquid

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Vehicle:

acetone/olive oil (4:1 v/v)

No. of animals per dose:

2 animals in Preliminary test
4 animals in main test

Details on study design:

The Preliminary Irritation/Toxicity Tests were performed in CBA/CaOlaHsd mice using two doses (2
animals/dose): 100% (undiluted) and 50% (w/v) in AOO. Based on the results, 50% (w/v) was selected
as top dose for the main tests.

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

- four groups of animals received MDAC (formulated in AOO) at 50, 25, 10 or 5% (w/v),

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

- a positive control group received 25 % (w/v) α-Hexylcinnamaldehyde (HCA), dissolved in AOO.

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.

Positive control substance(s):

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

Positive control results:

Positive control (25% (w/v) HCA in AOO gave a SI of 26.9

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

Negative (vehicle) control (AOO)

Key result

Parameter:

SI

Value:

6.2

Test group / Remarks:

MDAC 50% (w/v) in AOO

Key result

Parameter:

SI

Value:

4.2

Test group / Remarks:

MDAC 25% in AOO

Key result

Parameter:

SI

Value:

3.5

Test group / Remarks:

MDAC 10% (w/v) in AOO

Parameter:

SI

Value:

3.2

Test group / Remarks:

MDAC 5% (w/v) in AOO

Parameter:

SI

Value:

26.9

Test group / Remarks:

Positive control (25% (w/v) HCA in AOO )

Key result

Parameter:

SI

Value:

2.1

Test group / Remarks:

MDAC 50% (w/v)

During the Preliminary Irritation / Toxicity Test, the animals of the 100% (undiluted) were found dead on Day 2. No clinical signs were recorded for the animals of the 50% (w/v) dose group except of a very slight erythema (score 1) observed on Day 2 after treatment in both animals at 50% (w/v). No test item residual was observed on the ears of the animals. Clinical observations were summarized

Marked body weight loss (>5% reduction of body weight) was observed in one animal of the 50% (w/v) dose group on Day 6 (3 days after the last treatment) in the preliminary study. The body weight gains of the other animal in the 50% (w/v) dose group was normal and the mean body weight change was very slight (<5% reduction of body weights).

Ear thickness of the animals was measured by using a thickness gauge on Days 1, 3 and 6, and by ear punch weight determination after the euthanasia of the experimental animals on Day 6. The ear thickness values and the weights of the ear punches (2 per animal) were recorded. No increased ear thickness value (>25%) was detected. Ear punch weights of the animals of the 50% (w/v) dose group, were within the acceptable range.

The draining auricular lymph nodes of the animals were visually examined: they were normal in the 50% (w/v) dose group (subjective judgement by analogy with observations of former experiments).

Based on these results, the 100% (undiluted) group showed strong systemic toxicity, therefore it was not selected for the main test. However, the level of systemic toxicity of the 50% (w/v) dose group was considered acceptable and it was selected as top dose for the main test. Considering the marked body weight loss in one animal in the 50% (w/v) dose group during the observation period, an additional treatment group (5% (w/v) dose) was used in the first main assay.

Interpretation of results:

GHS criteria not met

Conclusions:

The test material showed no sensitisation potental (non-sensitizer) in this assay

Executive summary:

The object of this study was to determine the skin sensitisation potential of the test item following dermal exposure. 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 run to provide reliable information about the skin sensitisation potential of the test item for regulatory acceptance. The minimum number of animals was used, in accordance to the regulatory guidelines for animal welfare.

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 and the recommendations of the OECD No. 429 guideline, the best vehicle for the test item was Acetone: Olive oil 4:1 (v/v) mixture (AOO). The 100% (undiluted) test item was the highest concentration suitable for the test. The 50, 25, 10 and 5% (w/v) formulations appeared to be solutions by visual examination.

The Preliminary Irritation/Toxicity Tests were performed in CBA/CaOlaHsd mice using two doses (2 animals/dose): 100% (undiluted) and 50% (w/v) in AOO. Based on the results, 50% (w/v) was selected as top dose for the main tests.

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

- four groups of animals received MDAC (formulated in AOO) at 50, 25, 10 or 5% (w/v),

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

- a positive control group received 25 % (w/v) α-Hexylcinnamaldehyde (HCA), dissolved in AOO.

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.

In this experiment, there was no mortality or signs of systemic toxicity. No test item effect on body weight was observed. The calculated stimulation index values were 6.2, 4.2, 3.5, and 3.2 at concentrations of 50, 25, 10 and 5% (w/v), respectively.

However, the calculated results and the concluded positive (i.e. sensitizer) effect were not in line with the visual observation of lymph nodes. The SI value of the positive control substance was 26.9, this value was above the OECD 429 guideline criteria (being SI ≤ 20), and also outside the historical control range. Furthermore, the observed DPN values of the negative control samples were lower than anticipated (mean DPN value of the historical control range for this vehicle is 472.7 while in the performed experiment a mean DPN value of 189.1 was

noted (a value near the lower limit of the historical control range). Thus, there was a chance that the low negative control value in a study might cause a false positive study interpretation.

Based on these facts, an additional experiment was necessary in order to comply with the validity criteria of OECD No. 429 and provide reliable scientific conclusion. The second main assay was performed using the highest test item concentration and negative control group only (to minimize the number of animals used in the study):

- one group of animals received MDAC (formulated in AOO) at 50% (w/v),

- a negative control group received the vehicle (AOO) only.

In the second main assay, there was no mortality or signs of systemic toxicity. No test item effect on body weight was noted.

The calculated stimulation index value was 2.1 at concentrations of 50% (w/v), respectively.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

9 February 2017 - 4 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

This test is part of a tiered testing strategy for the evaluation of skin sensitisation potential

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name: MDAC
- Description: Colourless liquid
- CAS No. 13846-31-6
- Source of test material: Osaka Soda Co., Ltd.
- Batch No. of test material: 40201
- Expiration date of the batch: 26 January 2019
- Purity test date: 99.2%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protected from light
- Stability under test conditions: Assumed stable for the duration of the study
- Solubility and stability of the test substance in the solvent/vehicle: Soluble in Dimethyl Sulphoxide (DMSO) at 200 mM (Stock formulation concentration)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a stock liquid: 2000 µM. The test item was soluble in DMSO at 200 mM, on dilution of the stock formulation to a final concentration of 2000 μM in treatment culture medium a slight precipitate was observed.

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

The objective was to evaluate the potential of the test item, MDAC, to activate the Nrf2 transcription factor as part of a tiered strategy for the evaluation of skin sensitisation potential. Data generated with the present OECD 422D test guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment. This test cannot be used on its own to conclude on a skin sensitisation potential.

The test item was tested in two independent runs (each with cells from a different passage number). A third run was performed since non concordant results were obtained between the two first runs. A solubility assay was performed prior to the first treatment, the test item was found to be soluble in DMSO at 200 mM. This stock formulation was therafter diluted in treatment culture medium to the final concentration of 2000 µM.

Method for a run of 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 Dulbecco's Phosphate Buffered Saline (D-PBS) containing 0.05% Ethylenediamin-tetra-acetic-acid (EDTA). They were then harvested and re-suspended in maintanence medium no.2 and counted using Trypan Blue dye. The cell concentration was adjusted to a density of 8 x 10^4 cells/mL and distributed into four 96-well plates (three white and one transparent plate) by adding 125 µL (representing 1 x 10^4 cells) per well. After seeding, cells were grown for 24 (+/-1) hours in the 96 well microtiter plates prior to test item addition.

Treatment: After the 24 hour growing period, the medium was removed by aspiration and replaced with 150 µL of treatment medium. From the Master plate 4x, a volume of 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 evaporation and cross contamination between wells. Plates were then incubated for 48 (+/-2) hours at 37ºC, 5% CO2, 90% humidity.

Luminescence flash signal to evaluate induction signal - white plates: After incubation, supernatents from the white assay plates were discarded and the cells washed once with D-PBS. A 20 µL volume of passive lysis buffer was added to each well and incubated for 20 (+/- 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 the cytotoxicity: transparent plate: For the cell viability assay plate, the medium was replaced by 200 µL of treatment medium and 27 µL of MTT solution at 5 mg/ml in D-PBS was then added to each well of the transparent 96-well plate. Plates were covered with a sealing membrane and incubated at 37ºC in a humidified atmosphere for 4 hours (+/-10 minutes). At the end of incubation, the medium was removed and a volume of 200 µL of a 10% SDS solution was added to each well. The plates were covered with a sealing membrane and incubated at 37ºC in a humidified atmosphere overnight to extract the formazan from cells. After the overnight incubation, the absorption of each cell was deternined at 600 nm using a plate reader.

Positive control results:

Positive control: Cinnamic aldehyde (CA) (CAS No: 14371-10-9) stored at +4°C under nitrogen gas. Since several test items were assayed concurrently, the results of the positive control were shared.

For each run, the positive control item was dissolved in DMSO (final concentration 200 mM) and further diluted to a final concentration of 6.4 mM. It was diluted in DMSO by serial dilutions in the Master plate 100x, using a dilution factor of two, to obtain a total of five 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 formulations were prepared within 4 hours before use and kept at room temperature, protected from light until use.

In all three runs, all acceptance criteria were met for the positive controls whichnwere considered as valid.

Key result

Run / experiment:

other: Run 1: Evaluation criteria for a positive were partially met, considered negative. Run 2: All evaluation criteria met, considered positive. Run 3: The inductions were not statistically significant, evaluation criteria partially met, considered negative.

Parameter:

other: Keratinosens run

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

The first run was negative (EC1.5 > 1000 µM). The second run was positive (EC1.5 = 650.65 µM). As the results of the first two runs were not concordant, a third run was performed using a narrower range of concentrations. The third run was negative and therefore overall result was considered to be negative.

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: A slight precipitate was observed once the test item stock formulation was diluted in the treatment culture medium to a final concentration of 2000 μM.
Run 1: Concentrations: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM in culture medium containing 1% DMSO. A slight to moderate emulsion was observed in test item-treated wells at the end of the 48-hour treatment period at concentrations ≥ 1000 μM
Run 2: Concentrations: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM in culture medium containing 1% DMSO. A slight to moderate emulsion was observed in test item-treated wells at the end of the 48-hour treatment period at concentrations ≥ 1000 μM
Run 3: Concentrations: 45.67, 64.39, 90.79, 128.0, 180.5, 254.5, 358.9, 506.0, 713.5, 1006, 1418 and 2000 μM in culture medium containing 1% DMSO. A slight to moderate emulsion was observed in test item-treated wells at the end of the 48-hour treatment period at concentrations ≥ 1418 μM

Since emulsion was observed in test item-treated wells at the end of the 48-hour treatment period at concentrations ≥ 1000 μM, the luciferase activity may be underestimated at these concentrations.Therefore, the conclusion on the lack of activity cannot be drawn with sufficient confidence.

ACCEPTANCE CRITERIA
Each run was considered valid if the following criteria were met:
. the positive control results should be positive, thus the gene induction should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations,
. the average EC1.5 value for the positive control should be within 7 and 30 μM. In addition, the average induction (Imax) in the three replicate plates for the positive control at 64 μM should be between 2 and 8. If the latter criterion was not fulfilled, the dose-response of cinnamic aldehyde was carefully checked, and the run was accepted if there was a clear dose-response with increasing luciferase activity at increasing concentrations for the positive control,
. the average coefficient of variation of the luminescence reading in the negative control wells of the triplicate plates should be < 20%.

TEST ITEM EVALUATION CRITERIA
The results of each run are analyzed individually and if the test item is classified as positive in two runs,the final outcome is considered positive. If the test item is classified as negative in two runs, the final outcome is negative. In case, the first two runs were not concordant, a third run was performed and the final outcome was that of the two concordant runs.
The test item is considered as positive if the following four conditions are all met in two of two or in two of three runs, otherwise the KeratinoSens prediction is considered as negative:
. the Imax is > 1.5-fold and statistically significantly different as compared to the negative control (as determined by a two-tailed, unpaired Student’s T-test),
. at the lowest concentration with a gene induction > 1.5-fold (i.e. at the EC1.5 determining value), the cell viability is > 70%,
. the EC1.5 value is < 1000 μM (or < 200 μg/mL for test item without MW),
. there is an apparent overall dose-response for luciferase induction (or a reproducible biphasic response).
In cases of precipitate/emulsion in wells after the 48 (± 2) hours incubation period, luciferase activity may be underestimated and a negative conclusion cannot be drawn with sufficient confidence.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The average coefficient of variation of the luminescence reading in the negative control wells of the triplicate plates should be <20%.
- Acceptance criteria met for positive control: Gene induction should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations. The average EC1.5 value of the positive control should be within 7 and 30 µM. the average EC1.5 value for the positive control should be within 7 and 30 μM. In addition, the average induction (Imax) in the three replicate plates for the positive control at 64 μM should be between 2 and 8. If the latter criterion was not fulfilled, the dose-response of cinnamic aldehyde was carefully checked, and the run was accepted if there was a clear dose-response with increasing luciferaseactivity at increasing concentrations for the positive control.
Acceptance criteria met for variability between replicate measurements: Yes, with two exceptions: In the second run, the criterion "the average induction (Imax) in the three replicate plates for the positive control at 64 μM should be between 2 and 8" was not fulfilled (i.e. Imax of 8.48). However, since a clear dose-response with
increasing luciferase activity at increasing concentrations was obtained for the positive control, this was considered not to have any impact on the validity of the results of this run. In the third run, there was an invalidated experiment where the average coefficient of variability of the luminescence reading in the negative control wells of the triplicate plate was > 20% (i.e. 26.56). This run was repeated.

No geometric mean IC30 or IC50 was calculated since the cell viability was > 70% in all runs.

Interpretation of results:

GHS criteria not met

Remarks:

Part of the tiered strategy for skin sensitization assessment

Conclusions:

The test item MDAC was negative in the keratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor, though with limitations due to test item emulsion at concentrations ≥ 1000 µM.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The in vivo study (LLNA) confirms the test material is a non sensitisor and does not need classifying as such