Dimethyl heptanedioate

Endpoint summary

Description of key information

- In chemico study: direct peptide binding assay (DPRA, similar to 442C, GLP, K, Rel.1): predicted to be negative in the DPRA assay

- In vitro study: activation of keratinocytes (KeratinoSens, similar to 442D, GLP, K, Rel.1): predicted to be negative in the KeratinoSens assay

- In vitro study: activation of human cell line (h-CLAT, similar to 442E, GLP, K, Rel. 1): predicted to be negative in the h-CLAT assy

- Repiratory sensitisation: no data available

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 August 2017 - 27 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 442C and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted on 04 February 2015

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Remarks:

Certificate for GLP compliance was not attached to the study report to prove the GLP status.

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

Non-animal testing is the default requirement for skin sensitisation.

Details on the study design:

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 at the ratios 1:10 cysteine:test item and 1:50 lysine:test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with ultra-violet 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).

Preparation of test item and controls:
- Vehicle: acetonitrile (supplier: CARLO ERBA/batch number: P7E031027F) (without sonication)
- Positive control: Name : Cinnamaldehyde (CAS No.: 104-55-2, Batch No. : MKBX8146V, Supplier : Sigma-Aldrich), purity: 98.9%
- Co-elution control samples: In order to detect possible co-elution of the test item with a peptide, co-elution control samples were prepared by incubating the test item formulation with each buffer used to dilute the peptides. Cysteine or lysine peptides were not added to these samples.
- Reference control samples: All these control samples were prepared in triplicate and at the nominal concentration of 0.500 mM in the solvent (peptide solution cysteine or lysine). These samples were used to:
. reference control A: check the accuracy of the calibration curve for peptide quantification,
. reference control B: check the stability of the peptide during analysis,
. reference control C: check that the solvent did not impact the percentage of peptide depletion.
- Test item formulation preparation: The test item was pre-weighed and stored under appropriate conditions until ready to perform testing. It was dissolved in the selected vehicle (acetonitrile (supplier: CARLO ERBA/batch number: D6N042196N)) at 100 mM. This formulation was colorless limpid solution and was used just after its preparation.

Cysteine peptide:
. Peptide sequence : AC-RFAACAA-COOH
. Peptide sequence synonyms : AC-Arg-Phe-Ala-Ala-Cys-Ala-Ala-COOH ; 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
. Purity : 88.78%
. Expiry date : 07 February 2018
The cysteine peptide solution was freshly prepared at 0.667 mM in an aqueous phosphate buffer (pH 7.5) solution.

Lysine peptide
. Peptide sequence : AC-RFAAKAA-COOH
. Peptide sequence synonyms : AC-Arg-Phe-Ala-Ala-Lys-Ala-Ala-COOH; 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
. Purity : 96.92%
. Expiry date : 25 January 2018
The lysine peptide solution was freshly prepared at 0.667 mM in an aqueous ammonium acetate buffer (pH 10.2) solution.

Study design:
The test item was tested in one run per peptide. 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: In parallel, 250 μL of test item formulation was incubated with 750 μL of lysine peptide dilution buffer (without lysine peptide).

- Reference control A and B samples preparation
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 sample preparation
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: In parallel, 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).

- 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).

- Incubation of the samples
All samples (co-elution control, reference controls, test item and positive control samples) were incubated during 24 (± 2) hours at 25°C and protected from light before injection onto the HPLC/UV system. At the end of the incubation period, a visual inspection of the samples was performed prior to HPLC analysis to detect precipitate or phase separation. Samples presenting precipitate were centrifuged at 400g for a period of 5 minutes at room temperature and only supernatants were then injected onto the HPLC/UV system. Otherwise, the vials were directly transferred onto the HPLC/UV system.

- Preparation of the calibration curve samples
One set of calibration standards was prepared with each analytical sequence by spiking both peptides (lysine and cysteine) in separate 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. The calibration curves were defined by the relationships between the peak area of the peptide signal versus the nominal concentration. These curves were obtained by using the appropriate mathematical model.

- HPLC/UV analysis of the samples
The 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,
. one co-elution control sample,
. three reference control B samples,
. reference control C sample (replicate 1),
. positive control sample (replicate 1),
. test item study samples (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.

The HPLC/UV method used for the samples analysis according to the recommendations of the OECD guideline No. 442C is described in CiToxLAB France internal analytical method and is summarized in the table below:
. Analytical Column: Zorbax SB C18, 100 x 2.1 mm, 3.5 μm, (Waters) / In-line filter C18, 4.0 x 2.0 mm (Phenomenex)
. Mobil phase: Mobile phase A: acetonitrile + 0.085% TFA / Mobile phase B: milli-Q water + 0.1% TFA
. Flow: 350 µL/minute
. Gradient
Time % Mobile phase A % Mobile phase B
0 10 90
10 25 75
11 90 10
13 90 10
13.5 10 90
20 10 90
. UV Wavelength: 220 nm
. Rinse solution: Acetonitrile
. Oven temperature: 30.0°C
. Autosampler temperature: Nominal temperature of +25°C
. Injection volume: 5µL
. Retention times: Cysteine-peptide: approx. 9.8 minutes / Lysine-peptide: approx. 7.5 minutes
. Total analysis time: 20 minutes

Positive control results:

Cinnamic aldehyde was used as a positive control.
- Result of positive control samples in cysteine assay: Mean % depletion = 77.82% and SD = 0.13%
- Result of positive control samples in lysine assay: Mean % depletion = 50.29% and SD = 0.69%
Mean depletion rate of Cinnamaldehyde: 64.06%
The mean percent peptide depletion values for the positive control with its standard deviation value were within the acceptability criteria for the DPRA assay (cysteine and lysine reactivity assays).

Key result

Parameter:

other: Mean % depletion in Cysteine assay

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Value set to 0 due to negative depletion.

Key result

Parameter:

other: Mean % depletion in Lysine assay

Value:

0.59

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Parameter:

other:

Remarks:

Mean depletion rate

Value:

0.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No reactivity / minimal reactivity

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for reference controls: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Acceptance criteria met for the calibration curve: Yes
- Range of historical values if different from the ones specified in the test guideline: Not specified

Solubility results

The test item was found soluble at 100 mM in acetonitrile without sonication step. Therefore, this vehicle was retained.

Evaluation of the presence of precipitate at the end of the incubation with peptides

At the end of the incubation period, a visual inspection of all samples (co-elution controls, reference controls, test item and positive control samples) was performed prior to HPLC analysis.

As precipitate was observed in the test item samples incubated with the cysteine peptide, these vials were centrifuged at 400g for a period of 5 minutes at room temperature to force precipitate to the bottom of the vial. Positive control samples incubated with both peptides and reference control samples incubated with the cysteine peptide were also centrifuged at the same conditions to force precipitate to the bottom of the vial. Only supernatants were injected into the HPLC/UV system.

For the other samples (i.e. all the lysine samples: co-elution controls, reference controls and test item samples and co-elution controls prepared with cysteine peptide), the vials were directly transferred into the HPLC/UV system.

See the attached document "Tables of results DPRA"

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test item DIMETHYL PIMELATE, was considered to have no/minimal peptide reactivity, though with limitations due to its precipitation with the peptides.
The test item is considered negative in the DPRA assay.

Executive summary:

The skin sensitisation potential of the test item was evaluated using an in chemico direct peptide binding assay (DPRA) in accordance to the OECD Guideline No. 442C and in compliance with GLP. The reactivity of the test item was evaluated 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 at the ratios 1:10 cysteine:test item and 1:50 lysine:test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with ultra-violet 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 test item was dissolved at 100 mM in acetonitrile without sonication.

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.

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 using the formula described in § Data analysis and calculation:

. for the cysteine peptide, the mean depletion value was 0.00%,

. for the lysine peptide, the mean depletion value was 0.59%.

The mean of the percent cysteine and percent lysine depletions was equal to 0.30%. Accordingly, the test item was considered have no/minimal peptide reactivity. Therefore, the DPRA prediction is considered as negative and the test item may have no potential to cause skin sensitization.

Since precipitate was observed at the end of the incubation with the cysteine peptide, the peptide depletion may be underestimated. Therefore, the conclusion on the lack of reactivity could not be drawn with sufficient confidence.

However, since the mean of the cysteine percent and lysine percent depletions was < 6.38%, the test item was considered to have no or minimal peptide reactivity. Therefore, the DPRA prediction would be considered as negative and the test item is likely not to have any potential to cause skin sensitization, though with limitations due to its precipitation with the peptides.

This negative 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 of the test item.

Under the experimental conditions of this study, the test item DIMETHYL PIMELATE, was considered to have no/minimal peptide reactivity, though with limitations due to its precipitation with the peptides.

The test item is considered negative in the DPRA assay.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 September 2017 - 29 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 442D and in compliance with GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

Adopted on February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

Non-animal testing is the default requirement for skin sensitisation.

Details on the study design:

Test item formulations:
On the basis of solubility results, the test item was diluted in DMSO at 200 mM.
One formulation was prepared for each run. It was then diluted in DMSO by serial dilutions, using a dilution factor of 2 to obtain a total of 12 concentrations (0.98, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µM) 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 luciferase gene is the endpoint evaluated and reflects the activation by the test item of the Nrf2 transcription factor in this test
- Supplier: this cell line was provided by Givaudan
- Batch: C1 and D1
- Storage condition: at -80°C
- Mycoplasm: absence of mycoplasm was confirmed

Media:
- Maintenance medium No. 1: DMEM containing GlutaMAX^TM, 1000 mg/L D-Glucose, Sodium Pyruvate and supplemented with 9.1% Fetal Calf Serum (FCS) and 500 μg/mL G-418,
- maintenance medium No. 2: DMEM with 9.1% FCS without G-418,
- treatment medium: DMEM with 1% FCS without G-418,
- freezing medium: DMEM with 20% FCS and 10% DMSO.

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 10^4 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 10^4 cells) per well taking care to avoid sedimentation of the cells during seeding,
- after seeding, the cells were grown for 24 (± 1) hours in the 96-well microtiter plates prior to test item addition.

Preparation of the positive control:
The positive control used in the case of KeratinoSensTM is Cinnamic aldehyde.
For each run, the positive control item was dissolved in DMSO to a final concentration of 200 mM. This solution was then 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 these formulations were prepared within 4 hours before use, then kept at room temperature and protected from light until use.

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, a volume of 50 μL was added to each well of the three white assay plates and 50 μL to the transparent plate for the cytotoxicity evaluation,
- all plates were covered by a sealing membrane to avoid evaporation of volatile test items and to avoid cross-contamination between wells,
- the plates were then incubated for 48 (± 2) hours at 37°C, 5% CO2, 90% humidity.

Endpoint measurements:
- Microscopic observation
After the 48 (± 2) hours incubation period, the presence or absence of precipitate/emulsion was determined in each well by microscopic inspection.
- Luciferase activity measurement (96-well plate Luminometer with injectors and optical density reader (Varioskan Flash)):
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 (± 2) minutes at room temperature and 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.

Cytotoxicity assessment using MTT endpoint:
For the cell viability assay plate, the medium was replaced by 200 μL of treatment medium. A volume of 27 μL of a MTT solution at 5 mg/mL in D-PBS was then added to each well of the transparent 96-well plate, the plates were covered with a sealing membrane and returned at 37°C in the incubator in humidified atmosphere for 4 hours (± 10 minutes). At the end of the incubation period, 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 placed at 37°C in the incubator in 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.

Data analysis:
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 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 wrong extrapolations.
Also, the individual and overall geometric means IC50 and IC30 were calculated, when applicable.

Key result

Run / experiment:

other: First run

Parameter:

other: Imax

Remarks:

no EC1.5 calculated

Value:

1.13

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Second run

Parameter:

other: Imax

Remarks:

no EC1.5 calculated

Value:

1.12

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Microscopic observation: no precipitate/emulsion was observed in any test item-treated-wells at the end of the 48-hour treatment period in either run
- Cell viability: no noteworthy decrease in cell viability was noted and no IC30 or IC50 was calculated (i.e. cell viability > 70%) in both runs

__TBC__
ACCEPTANCE OF RESULTS:
- The luciferase activity induction obtained with the positive control, Cinnamic Aldehyde, was above the threshold of 1.5-fold in at least one concentration.
- The EC1.5 of the positive control was within two standard deviations of the historical mean [0.8 - 27.1] (first run: 4.29 µM ; second run:8.22 µM).
- The average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (first run: 17.12% ; second run: 9.86%).

Table 1. Evaluation of the viability (%) of cultures treated with the test item for each run

	Concentration (µM)
Dimethyl pimelate	0.98	1.95	3.91	7.81	15.63	31.3	62.5	125	250	500	1000	2000
Viability (%) in Run 1	93	102	96	104	97	96	94	93	97	91	88	78
Viability (%) in Run 2	105	103	101	105	103	102	110	103	100	95	99	96
Mean viability (%)	99	102	98	105	100	99	102	98	99	93	93	87
Geometric Mean (%)	99	102	98	105	100	99	102	98	99	93	93	87
SD	8	1	4	1	4	4	12	7	2	3	8	12

Table 2. Gene induction values, Imax, IC30, IC50 and EC1.5 values, mean and SD values obtained after treatment with the test item in each run

	Concentration (µM)
Dimethyl pimelate	0.98	1.95	3.91	7.81	15.63	31.3	62.5	125	250	500	1000	2000
Induction values in Run 1	0.9	1.0	1.0	1.0	0.9	1.1	0.8	0.8	0.8	0.7	0.7	0.8
Induction values in Run 2	1.1	1.1	1.1	1.1	1.0	1.0	1.0	1.1	1.1	1.0	0.9	0.9
Mean induction	1.0	1.0	1.1	1.0	1.0	1.1	0.9	0.9	0.9	0.8	0.8	0.9
SD	0.1	0.1	0.1	0.1	0.0	0.1	0.1	0.2	0.2	0.1	0.1	0.1

Table 3. Imax and EC1.5 results

Dimethyl pimelate	Imax	EC1.5 (µM)	IC50 (µM)	IC30 (µM)
Run 1	1.13	-	-	-
Run 2	1.12	-	-	-
Mean	1.12	n.r.	n.r.	n.r.
Geometric Mean	n.r.	-	-	-
SD	0.01	-	-	-

- : no data available

n.r.: not requested by the OECD Guideline

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test item, DIMETHYL PIMELATE, was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.

Executive summary:

The test item was evaluated for the ability to activate the Nrf2 transcription factor in a KeratinoSens(TM) test similar to the OECD Guideline No. 442D and in compliance with GLP. In this test, KeratinoSens(TM) cells cultures were treated with 12 concentrations of test chemical (0.98, 1.95, 3.91, 7.81, 15.63, 62.5, 125, 250, 500, 1000 and 2000 µM) and control substances (positive control: Cinnamic Aldehyde in DMSO, vehicle control: 1% DMSO in exposure medium, blank) and were then incubated for about 48 hours at 37 +/-1.0°C in presence of 5% CO2. Luciferase activity (Imax and EC1.5) was assessed using a luminometer. The toxicity of the test substance was evaluated with the assessment of cell viability (MTT test). A total of 2 independent experiments were performed.

All acceptance criteria were met for the positive and negative controls in both runs; they were therefore considered as validated.

Both runs were performed using the following concentrations; 0.98, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 μM 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-hour treatment period in either run,

. no noteworthy decrease in cell viability was noted and no IC₃₀or IC₅₀was calculated (i.e.cell viability > 70%) in both runs,

. nostatistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations, in either run,

. moreover, the I_maxvalues were<1.5 in both runs (i.e.1.13 and 1.12 in the first and second runs, respectively) and no EC_1.5was calculated in either run.

The evaluation criteria for a negative response are met in both runs, the final outcome is therefore negative.This negative 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 lack of skin sensitization potential.

Under the experimental conditions of this study, the test item, DIMETHYL PIMELATE, was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 December 2017 - 26 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Study performed according to a protocol similar to the OECD test guideline No. 442E and in compliance with GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

yes

Remarks:

Reactivity check with both PC (NiSO4 and DNCB) instead of one (DNCB). Cell preparation: cells seeded between 0.1-0.2 x 10^6 cells/mL for 48-72h instead of the opposite. Dose finding assay: top concentration tested was 5000 µg/mL with DMSO as solvent.

Qualifier:

equivalent or similar to guideline

Guideline:

other: DB-ALM Protocol No. 158: human Cell Line Activation Test (h-CLAT)

GLP compliance:

yes

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

Non-animal testing is the default requirement for skin sensitisation.

Details on the study design:

TEST ITEM AND CONTROLS PREPARATION

Positive controls preparation
The positive control, DNCB, was prepared at the concentration of 8 µg/mL in DMSO as follows:
- on the treatment day, the required quantity of DNCB was mixed with DMSO at the concentration of 2 mg/mL,
- this solution was then 250-fold diluted in cRPMI in order to obtain a 8 µg/mL DNCB stock solution.

The positive control NiSO4 was prepared at the concentration of 200 µg/mL in 0.9% NaCl as follows:
- on the treatment day, the required quantity of NiSO4 was mixed with 0.9% NaCl at the concentration of 10 mg/mL,
- this solution was then 50-fold diluted in cRPMI in order to obtain a 200 µg/mL NiSO4 stock solution.
Both positive control stock solutions were prepared within 4 hours before use, and kept at room temperature and protected from light until use.

Vehicle control preparation
As DMSO was the vehicle selected at completion of the solubility assay, DMSO control formulation was included as vehicle control, and consisted in DMSO dissolved at 0.2% in cRPMI.

Test item preparation
All test item preparations were prepared in glass vials only. Test concentrations prepared and vehicle used were indicated by the Study Director in the study files, and no study plan amendment was issued for these purposes.
Fresh stock formulations of the test item were prepared for each run, using the vehicle and concentration identified in the § Solubility assessment. These concentrations were the same for all runs.
Test item formulations prepared in DMSO were 500 x concentrated; then 2 x concentrated formulations were prepared by 1:250 dilution in cRPMI. A DMSO vehicle control was also prepared (0.4% DMSO in cRPMI). The above mentioned dilutions of the test item and vehicle controls were performed to insure a constant percentage of the vehicle in the final volume of cell suspension in the well (i.e. 0.2% for DMSO).
The aspect of the stock formulations was evaluated and recorded in the study files.
The precipitation in the treatment conditions (i.e. when diluted in cRPMI) was checked and any observation was reported in the study files.
The test item formulations were kept at room temperature and protected from light until use, i.e. within 4 hours after preparation of the stock formulations. No control of concentration was performed during the study.


TEST SYSTEM

- Cells
The THP-1 is an immortalized human monocytic leukemia cell line derived from an acute monocytic leukemia patient. The THP-1 cell line is obtained from ATCC (Ref: TIB-202, American Type Culture Collection, Manassas, USA) by the intermediate of LGC Standards (Molsheim, France).
The THP-1 cells are stored in a cryoprotective medium in a liquid nitrogen container.
Cells were grown using general culture procedures. They were cultured in cRPMI medium and maintained in a humidified incubator set at 37°C, 5% CO2 and were not allowed to exceed a cell density of 1 x 10^6 cells/mL or more than 30 passages.
The culture medium (cRPMI) was composed of RPMI 1640 with 10% FBS, 0.05 mM 2 mercaptoethanol and with penicillin and streptomycin.
During cell culturing, cell viability was checked using trypan blue.

- Cell culture for testing
For testing, THP-1 cells were seeded at a density between 0.1 x 10^6 cells/mL and 0.2 x 10^6 cells/mL, and pre-cultured in culture flasks for 48 hours to 72 hours, respectively. Cell did not exceed density of 1 x 10^6 cells/mL. On the day of testing, cells harvested from culture flasks were resuspended with fresh culture medium at 2 x 10^6 cells/mL. Then, 500 µL of cells suspension were distributed into a 24 well flat bottom plate (i.e. 1 x 10^6 cells/well).

- Reactivity check
Two weeks after thawing, a reactivity check was performed to qualify the cells of each working cell bank before testing. A reactivity check assay was performed by testing cell response after contact with Lactic Acid, DNCB and NiSO4 under an internal Citoxlab France reference number.
Results from reactivity check tests are compiled in Citoxlab France internal data, with negative and positive control data obtained during each test.


STDUDY DESIGN

The study was divided in two successive phases.
First, a Dose-Range Finding assay (DRF) was performed to assess test item toxicity and, determine the CV75 i.e. the test item concentration that result in 75% cell viability compared to the vehicle control. Secondly, based on cytotoxicity data obtained from the DRF, a concentration series was tested in successive runs in the main tests.
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: 7.81, 15.63, 31.25, 62.50, 125.00, 250.00, 500.00 and 1000.00 µg/mL.
Each assay was performed as described here below.

Test item stock solutions were prepared at eight different concentrations by 2-fold dilutions using the selected vehicle. These stock formulations were then diluted 250-fold (as DMSO is the selected vehicle) 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 then 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 three validated 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 was obtained. The maximum concentration in the plates was 361.16 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 as noted in § Test item and controls preparation.
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 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): 361.16, 300.97, 250.81, 209.00, 174.17, 145.14, 120.95, 100.79 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.


FLOW CYTOMETRY ANALYSIS

- DRF assays
The PI uptake is analyzed using flow cytometry with the acquisition channel B3. A total of 10 000 living cells (PI negative) are acquired. In case of low viability which does not allow obtaining 10 000 living cells, a total of 30 000 events is acquired. Alternatively, cells were acquired for a maximum of 1 minute after the initiation of the acquisition.

- Main test
The non-specific binding of IgG1 and the expression CD86 and CD54 was analyzed by flow cytometry with the acquisition channel B1 in order to obtain the Mean Fluorescence Intensity (MFI); whereas the viability (PI uptake) was analyzed with the acquisition channel B3. A total of 10 000 living cells (PI negative) were acquired. When the viability was low and did not allow obtaining 10 000 living cells, a total of 30 000 events was acquired. Alternatively, cells were acquired for a maximum of 1 minute after the initiation of the acquisition.
In case cell viability is less than 50%, no MFI is presented in the study report and the corresponding test item concentration are considered too high for interpretation because of the diffuse labelling cytoplasmic structures that are generated following cell membrane destruction.


CALCULATIONS

- Estimation of the CV75 value
The percentage of living cells (PI negative cells) is used as the value for cell viability. The CV75 value is derived from the dose-response curve as shown in Figure 1 (75% of cell viability, lying between a and c) (section Attached background material). CV75 is defined as the estimated concentration that is required to elicit 75% cell viability. The CV75 value is calculated by log-linear interpolation utilizing the following equation:
Log CV75= [(75 - c) x Log b - (75 - a) x Log d] / (a - c)

- Main test
Based on the Mean Fluorescence Intensity (MFI), the Relative Fluorescence Intensity (RFI) of CD86 and CD54 were calculated according to the following equation:
RFI= [MFI of the test item-treated (CD86 or CD54) - MFI of test item-treated IgG1] / [MFI of control-treated (CD86 or CD54) - MFI of control-treated IgG1] x 100
RFI = Relative Fluorescence Intensity
MFI = Mean Fluorescence Intensity


ACCEPTANCE CRITERIA

- DRF:
*Viability of control cells treated with cRPMI (and DMSO if applicable) should be ≥ 90%,
*viability of control cells treated with 0.2% DMSO should be ≥ 90%, if applicable.

- Main test
The following applies for each run.
*Controls acceptance criteria
- Viability of cells treated with cRPMI and DMSO controls should be ≥ 90%,
- in cRPMI and DMSO control wells, MFI ratio of both CD86 and CD54 to isotype control should be > 105%,
- in the DMSO control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI > 150% and CD54 RFI ≥ 200%),
- in the positive controls (DNCB and NiSO4), RFI values of both CD86 and CD54 should meet positive criteria (CD86 RFI ≥ 150 and CD54 RFI ≥ 200) and cell viability should be more than 50%.
*Test item acceptance criteria
- For a test item noted as cytotoxic in the DRF phase, and resulting in a negative outcome in the main test, cell viability at 1.2 x CV75 should be < 90% in each run,
- cell viability of at least 4 out of 8 concentrations should be > 50%.

- Main test interpretation
*Individual run interpretation
A run conclusion is positive if at least one of the conditions below is met:
- RFI of CD86 is ≥ 150 at any concentration leading to ≥ 50% viability,
- RFI of CD54 is ≥ 200 at any concentration leading to ≥ 50% viability.
In other circumstances, the run is considered as negative.

*Prediction model
Based on the individual run conclusions, a final prediction is made as follows (see Figure 2, section Attached background material):
- if the first two runs are both positive for CD86 and/or are both positive for CD54, the h-CLAT prediction is considered POSITIVE and a third run does not need to be conducted,
- if the first two runs are negative for both markers, the h-CLAT prediction is considered NEGATIVE (with due consideration of the highest-tested dose conditions) without the need for a third run,
- if however, the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 (hereinafter referred to as P1) and the other is only positive for CD54 (hereinafter referred to as P2), a third run is required. If this third run is negative for both markers (hereinafter referred to as N), the h-CLAT prediction is considered NEGATIVE. On the other hand, if the third run is positive for either marker (P1 or P2) or for both markers (hereinafter referred to as P12), the h-CLAT prediction is considered POSITIVE.


CLASSIFICATION

Results from the present study can be used to support the discrimination between skin sensitizers (i.e. UN GHS Category 1) and non-sensitizers in the context of Integrated Approaches to Testing and Assessment (IATA). However, results obtained at completion of the study will not be usable on their own, neither to sub-categorize skin sensitizers into sub-categories 1A and 1B as defined by UN GHS, for authorities implementing these two optional sub-categories, nor to predict potency for safety assessment decisions.

Positive control results:

Results of positive controls met the acceptance criteria defined for this method. Furthermore, classification of these controls were verified by the Reactivity Check data (provided in appendix of the study report).

Key result

Run / experiment:

other: General conclusion

Parameter:

other: Relative Fluorescence Index (RFI)

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Post-treatment observations: no precipitate/emulsion was noted in treated wells.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes (Reactivity check data)

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Results of the solubility assessment

Results obtained from the solubility assay are summarized in the table below:

Vehicle	Concentration (mg/mL)	Aspect	Retained vehicle
0.9% NaCl	100	Insoluble, two separated phases	No
DMSO	500	Colorless homogenous solution	Yes

Therefore, DMSO was selected as vehicle, and the following test item concentrations were tested in the DRF phase: 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 µg/mL.

Dose-Range Finding results

The following results were obtainedin the first DRF test (i.e. DRF 1):

- at post-treatment observation, emulsion was noted at concentration ≥ 7.81 µg/mL,

- flow cytometry measurement after Propidium Iodide staining revealed a cell viability decrease below 75% at concentrations ≥ 500 µg/mL. The corresponding CV75 value was 276.04 µg/mL.

 

The following results were obtainedin the second DRF test (i.e. DRF 2):

- at post-treatment observation, emulsion was noted at concentration ≥ 7.81 µg/mL,

- flow cytometry measurement after Propidium Iodide staining revealed a cell viability decrease below 75% at concentrations ≥ 500 µg/mL. The corresponding CV75 value was 325.91 µg/mL.

 

Based on the results from both DRF runs, the mean CV75 was 300.97 µg/mL, and the highest concentration tested in the main test was therefore 361.16 µg/mL (i.e.1.2-fold the mean CV75).

Main test summary results

Test item name	Conc. (µg/mL)	RFI for CD86			RFI for CD54			Viability (%)			Run conclusion			General conclusion
		A	B	C	A	B	C	A	B	C	A	B	C
Dimethyl Pimelate	100.79	132	101	94	295	160	122	92.6	93.6	93.2	P12	N	N	Negative
	120.95	128	98	87	285	152	128	91.2	92.0	92.6
	145.14	119	89	82	300	158	132	89.6	90.8	93.3
	174.17	153	92	85	225	133	119	89.9	91.8	91.5
	209.00	127	91	78	235	131	135	83.9	91.1	88.9
	250.81	123	88	81	175	119	115	78.1	88.3	88.1
	300.97	85	75	66	160	90	104	70.2	79.3	83.0
	361.16	79	55	51	-25	60	71	50.7	67.8	70.2

N= run with negative outcome

P₁= run with positive outcome for CD86

P₂= run with positive outcome for CD54

P₁₂= run with positive outcome for CD86 and CD54

Conc.= concentration

RFI= Relative Fluorescence Index

All acceptance criteria were reached in each run.

 

Results from each run are summarized below.

 Run A:

-           Post-treatment observations: no precipitate/emulsion was noted in treated wells.

-           Run outcome:

- RFI CD86 exceeded the positivity threshold at the concentration of 174.17,

- RFI CD54 exceeded the positivity threshold at concentrations ≤209.

=> The run was therefore considered positive.

Run B:

-           Post-treatment observations: no precipitate/emulsion was noted in treated wells.

-           Run outcome: RFI CD86 and RFI CD54 did not exceed the positivity thresholds at any tested concentration.

=> The run was therefore considered negative.

 

Run C:

-            Post-treatment observations: no precipitate/emulsion was noted in treated wells.

-            Run outcome: RFI CD86 and RFI CD54 did not exceed the positivity thresholds at any tested concentration.

=> The run was therefore considered negative.

As a general conclusion according to the prediction model (figure 2) used in the h-CLAT test method, the test is considered as negative.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test item, DIMETHYL PIMELATE, was found to be negative in the h-CLAT.

Executive summary:

The ability of the test item to induce an increase in cell surface markers expression in THP-1 cells was evaluated using the h-CLAT test method in accordance to the OECD Guideline No. 442E and in compliance with GLP.

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. The test item was found soluble in DMSO at the concentration of 500 mg/mL.

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 test item induced a decrease in cell viability < 75% in both DRF runs and the calculated mean CV75 value was: 300.97 µg/mL. Therefore, the highest concentration tested in the main test was 361.16 µg/mL (i.e.1.2-fold the mean CV75).

The skin sensitizing potential of the test item was then tested in the main test in three 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 labelled with IgG1-FITC antibodies, the second one was labelled 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).

All acceptance criteria were reached in each run and no precipitate/emulsion was noted in treated cells.

In the three successive runs, two of them were negative and one was considered positive (RFI CD86 exceeded the positivity threshold at the concentration of 174.17 µg/mL and, RFI CD54 exceeded the positivity threshold at concentrations ≤ 209 µg/mL). Therefore, according to the prediction model followed for the h-CLAT test method, the test is considered to be negative.

Under the experimental conditions of this study, the test item, DIMETHYL PIMELATE, was found to be negative in the h-CLAT and therefore was considered to have no potential to induce an increase in cell surface markers expression in THP-1 cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Three in chemico/in vitro key studies were identified :

- In the in chemico assay (Michel, 2017, rel.1), the skin sensitisation potential of the test item was evaluated using a direct peptide binding assay (DPRA) similarly to the OECD Guideline No. 442C and in compliance with GLP. The skin sensitisation potential of the test item was evaluated using an in chemicodirect peptide binding assay (DPRA) in accordance to the OECD Guideline No. 442C and in compliance with GLP.The reactivity of the test item was evaluated 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 at the ratios 1:10 cysteine:test item and 1:50 lysine:test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with ultra-violet 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 acceptance criteria of the samples for the calibration curve, QC and positive control were satisfied. The study was therefore considered to be valid.

The mean of the percent cysteine and percent lysine depletions was equal to 0.30%. Since precipitate was observed at the end of the incubation with the cysteine peptide, the peptide depletion may be underestimated. Therefore, the conclusion on the lack of reactivity could not be drawn with sufficient confidence. However, since the mean of the cysteine percent and lysine percent depletions was < 6.38%, the test item was considered to have no or minimal peptide reactivity.

Therefore, the DPRA prediction is considered as negative and the test item may have no/minimal peptide reactivity, though with limitations due to its precipitation with the peptides.

- In the in vitro study (Richez, 2018, rel. 1), the test item was evaluated for the ability to activate the Nrf2 transcription factor in a KeratinoSens(TM) test similar to the OECD Guideline No. 442D and in compliance with GLP. In this test, KeratinoSens(TM) cells cultures were treated with 12 concentrations of test chemical (0.98, 1.95, 3.91, 7.81, 15.63, 62.5, 125, 250, 500, 1000 and 2000 µM) and control substances (positive control: Cinnamic Aldehyde in DMSO, vehicle control: 1% DMSO in exposure medium, blank) and were then incubated for about 48 hours at 37 +/-1.0°C in presence of 5% CO2. Luciferase activity (Imax and EC1.5) was assessed using a luminometer. The toxicity of the test substance was evaluated with the assessment of cell viability (MTT test). A total of 2 independent experiments were performed.

All acceptance criteria were met for the positive and negative controls in both runs; they were therefore considered as validated.

At the tested concentrations:

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

- no noteworthy decrease in cell viability was noted and no IC₃₀ or IC₅₀ was calculated (i.e.cell viability > 70%) in both runs,

- nostatistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations, in either run,

- moreover, the I_max values were<1.5 in both runs (i.e. 1.13 and 1.12 in the first and second runs, respectively) and no EC_1.5 was calculated in either run.

The evaluation criteria for a negative response are met in both runs, the final outcome is therefore negative.

Under the experimental conditions of this study, the test item, DIMETHYL PIMELATE, was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.

- In the in vitro study (Gerbeix, 2018, rel. 1), the ability of the test item to induce an increase in cell surface markers expression in THP-1 cells was evaluated using the h-CLAT test methodin accordance to the OECD Guideline No. 442E and in compliance with GLP.

A solubility assessment was first performed in which the test item was found soluble in DMSO at the concentration of 500 mg/mL. Then 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 test item induced a decrease in cell viability < 75% in both DRF runs and the calculated mean CV75 value was: 300.97 µg/mL. Therefore, the highest concentration tested in the main test was 361.16 µg/mL (i.e.1.2-fold the mean CV75).

The skin sensitizing potential of the test item was then tested in the main test in three 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 labelled with IgG1-FITC antibodies, the second one was labelled 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).

All acceptance criteria were reached in each run and no precipitate/emulsion was noted in treated cells.

In the three successive runs, two of them were negative and one was considered positive (RFI CD86 exceeded the positivity threshold at the concentration of 174.17 µg/mL and, RFI CD54 exceeded the positivity threshold at concentrations ≤ 209µg/mL). Therefore, according to the prediction model followed for the h-CLAT test method, the test is considered to be negative.

Under the experimental conditions of this study, the test item, DIMETHYL PIMELATE, was found to be negative in the h-CLAT and therefore was considered to have no potential toinduce an increase in cell surface markers expression in THP-1 cells.

Based on the results of these three in chemico/in vitro tests, the test item, DIMETHYL PIMELATE, cannot be considered as a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self classification:

Based on the available information, the substance should not be classified as skin sensitizer according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the UN-GHS.