(S)-N,N'-dibutyl-2-[(1-oxododecyl)amino]glutaramide

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 December 2016 to 09 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.40.Bis: “In Vitro Skin Corrosion: Human Skin Model Test"

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(S)-N,N'-dibutyl-2-[(1-oxododecyl)amino]glutaramide

EC Number:

264-391-1

EC Name:

(S)-N,N'-dibutyl-2-[(1-oxododecyl)amino]glutaramide

Cas Number:

63663-21-8

Molecular formula:

C25H49N3O3

IUPAC Name:

(2S)-N,N'-dibutyl-2-dodecanamidopentanediamide

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EPISKIN™(SM) is a three-dimensional human skin model.

Justification for test system used:

The EPISKIN™(SM) model has been validated for corrosivity testing in an international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™(SM), supplied by SkinEthic, France.
- Tissue batch number: 16-EKIN-049
- Expiry date: 12 December 2016
- Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- The EPISKIN™(SM) kits were kept in their packaging at 37 °C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8 °C until the initiation of the test.
- In each case, the pH of the agar medium used for transport was checked by checking the colour of the medium: orange colour = good or yellow or violet colour = not acceptable. The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40 °C (the colour change is irreversible, independent of the length of the period above 40 °C): white colour = good or grey or black colour = not acceptable. The kits were found to be in good order at reception.

CHECK FOR DIRECT MTT REDUCTION
Approximately 20 mg of test material was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37°C in an incubator with 5 % CO2, in a >95 % humidified atmosphere for 3 hours and then any colour change was observed: Test materials which do not react with MTT: yellow or Test materials reacting with MTT: blue or purple. After three hours incubation, yellow colour of the mixture was detected; therefore additional controls were not used in the experiment.

CHECK TO DETECT COLOURING POTENTIAL OF THE TEST MATERIAL
Prior to treatment, the test material was evaluated for its intrinsic colour or ability to become coloured in contact with water and/or isopropanol. As the test material had an intrinsic colour, thus further evaluation to detect colouring potential was necessary. Non Specific Colour % (NSCliving %) was determined in order to evaluate the ability of test material to stain the epidermis by using additional control tissues.
Therefore, in addition to the normal procedure, two additional test material-treated living tissues were used for the non specific OD evaluation. These tissues followed the same test material application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test material that may be present in the test disks. OD readings were conducted following the same conditions as for the other tissues.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (22.9 to 24.2 °C)
- Temperature of post-treatment incubation: 37 °C (with MTT)

PRE-INCUBATION (DAY -1)
The Maintenance Medium was pre-warmed to 37 °C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37 °C in an incubator with 5 % CO2 in a > 95 % humidified atmosphere.

APPLICATION (DAY 0)
-The Assay Medium was pre-warmed to 37 °C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, whereby each epidermis was in contact with the medium in the corresponding well underneath. Two epidermis units were used for each test or control materials.
- 20 mg of test material was applied evenly to the epidermal surface of each of two test material treated units and each additional control skin units and then 100 μL physiological saline was added to the test material to ensure good contact with the epidermis.
- 50 μL of physiological saline was added to each of the two negative control skin units.
- 50 μL of glacial acetic acid was added to each of the two positive control skin units. The plates with the treated epidermis units were incubated for 4 hours (± 10 min) at room temperature (22.9 to 24.2 °C) covered with the plate lids.

REMOVAL OF TEST MATERIAL AND CONTROLS
After the incubation time (4 hours), all test material treated tissues or also the positive control tissues were removed and rinsed thoroughly with PBS solution to remove all the remaining test or positive control material from the epidermal surface. Likewise, negative control tissues were processed accordingly. The rest of the PBS was removed from the epidermal surface using a pipette (without touching the epidermis).

MTT TEST/ FORMAZAN EXTRACTION (DAY 0)
MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units (except of the two living colour control units). The lid was replaced and the plate incubated at 37 °C in an incubator with 5 % CO2 for 3 hours (± 15 minutes), protected from light.
At the end of incubation with MTT a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this procedure involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated overnight at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.
A blank sample containing 2 mL of acidified isopropanol was processed in parallel.

CELL VIABILITY MEASUREMENTS (DAY 1)
Following the formazan extraction, 2× 200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as the blank.

NUMBER OF REPLICATE TISSUES: 2

CALCULATIONS OF VIABILITY PERCENTAGES
The data calculation using two replicates is shown below. Results were calculated in a similar way when more replicates are used.
-Blank: The mean of the 6 blank OD values was calculated

-Negative control: Individual negative control OD values (NCraw) were corrected with the mean blank OD:
OD Negative Control (ODNC) = ODNCraw – ODblank mean.
The corrected mean OD of the 2 negative control values was also calculated: this corresponds to 100 % viability

-Positive control: Individual positive control OD values (PCraw) were corrected with the mean blank OD:
OD Positive Control (ODPC) = ODPCraw – ODblank mean.
The corrected mean OD of the 2 positive control values was calculated, the % viability for each positive control replicate was calculated relative to the mean negative control:
Positive Control1 % = (ODPC1 / mean ODNC) ×100
Positive Control2 % = (ODPC2 / mean ODNC) ×100
The mean value of the 2 individual viability % for positive control was calculated:
Mean PC % = (PC1 % + PC2 % ) / 2

-Test material: Individual test material OD values (TTraw) were corrected with the mean blank OD:
OD Treated Tissue (ODTT) = ODTTraw – ODblank mean.
The corrected mean OD of the 2 test material values was calculated. The % viability for each test material replicate was calculated relative to the mean negative control:
Treated Tissue1 % = (ODTT1 / mean ODNC) ×100
Treated Tissue2 % = (ODTT2 / mean ODNC) ×100
The mean value of the 2 individual viability % for test material was calculated:
Mean TT % = (TT1 % + TT2 %) / 2.
The variability for 2 disks was calculated as:
(Disk1-Disk2)/((Disk1+Disk2)/2) x 100 %

-Data calculation for test materials having MTT-interacting potential
Test materials that interfere with MTT can produce non specific reduction of the MTT. In this case, additional control samples are used to determine the OD value derived from non-specific reduction of the MTT. The measured OD value is corrected by the result of the additional controls before calculation of viability % as follows:
Non specific MTT reduction calculation:
NSMTT% = [(ODKT- ODKNC) / ODNC] × 100
ODKNC: negative control treated killed tissues OD
ODKT: test material treated killed tissues OD
ODNC: negative control OD
If NSMTT % is ≤ 50 %, then true MTT metabolic conversion (TODTT) has to be undertaken as follows:
TODTT = [ODTT – (ODKT – ODKNC)]
ODTT: test material treated viable tissues
The % relative viability (RV %) for each test material replicate is calculated relative to the mean negative control:
RV1 % = [TODTT1 / mean ODNC] × 100
RV2 % = [TODTT2 / mean ODNC] × 100
The mean value of the 2 individual relative viability % for test material is calculated = (RV1 % + RV2 %) / 2
If NSMTT % is > 50 % relative to the negative control: additional steps must be undertaken if possible, or the test material must be considered as incompatible with the test.

- Data calculation for test materials having colouring potential
For test materials detected as able to stain the tissues the non specific OD was evaluated due to the residual chemical colour (unrelated to mitochondrial activity) and subtracted before calculation of the “true” viability % as detailed below:
Non Specific Colour % NSCliving % = (mean ODCTV / mean ODNC)×100
ODCTV: test material treated viable tissue (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)
If NSC living % is ≤ 5 % then the normal calculation mode was used.
If NSC living % is > 5 % and ≤ 5 0 %, then additional correction (TODTT) has to be undertaken as follows:
TODTT = [ODTV - ODCTV]
ODTT: test material treated viable tissue (incubated with MTT)
ODCTV: test material treated viable tissue (not incubated with MTT)
The % relative viability (RV% %) for each test material replicate is calculated relative to the mean negative control:
RV1 % = [TODTT1 / mean ODNC] × 100
RV2 % = [TODTT2 / mean ODNC] × 100
The mean value of the 2 individual relative viability % for test material is calculated:
Mean Relative Viability % = (RV1 % + RV2 %) / 2
If NSC living % is > 50 % relative to the negative control, additional steps must be undertaken if possible, or the test material must be considered as incompatible with the test.

-Data calculation for test materials having both MTT-interacting and colouring potential
For test materials detected as able to both stain the tissues and interfere with MTT may also require a third set of controls before calculation of the “true” viability %.
Non Specific Colour % with killed tissues NSCkilled % = (mean ODCTK / mean ODNC)×100
ODCTK: test material treated killed tissues (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)
TODTT = [ODTT – (ODKT – ODKNC) – mean ODCTV +mean ODCTK]
ODTT: test material treated viable tissues (incubated with MTT)
ODKT: test material treated killed tissues OD
ODKNC: negative control killed tissues OD
ODCTV: test material treated viable tissues (not incubated with MTT)
ODCTK: test material treated killed tissues (not incubated with MTT)
The % relative viability (% RV) for each test material replicate is calculated relative to the mean negative control:
RV1 % = [TODTT1 / mean ODNC] × 100
RV2 % = [TODTT2 / mean ODNC] × 100
The mean value of the 2 individual relative viability % for test material is calculated: Mean Relative Viability % = (RV1 % + RV2 %) / 2

INTERPRETATION OF TEST RESULTS
For 2 disks: If both disks have mean viability of ≥ 35 % = Non Corrosive, If both disks have mean viability of < 35 % = Corrosive (at the corresponding incubation period).
Otherwise:
If the mean value is ≥ 35 % and the variability is less than 50 % = Non Corrosive.
If the mean value is <35 % and the variability is less than 50 % = Corrosive.
Otherwise: If the classification is not made with these criteria, retest with 2 more disks. Take the mean of the 4 disks to classify as above or below 35 %. Outlier values may be excluded where there are scientific reasons, such as where application or rinsing is difficult and that the Study Director considers that a result is not representative.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 20 mg (+ 100 µL of physiological saline to ensure good contact with the epidermis)

NEGATIVE CONTROL
- Amount(s) applied: 50µL
- Concentration: 0.9 % w/v

POSITIVE CONTROL
- Amount(s) applied: 50 µL

Duration of treatment / exposure:

4 hours ± 10 minutes

Duration of post-treatment incubation (if applicable):

3 hours ± 15 minutes (with MTT)

Number of replicates:

The test was performed in duplicate.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

99.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS: ADDITIONAL CONTROLS
- Colour interference with MTT: As the test material was coloured, two additional test material-treated tissues were used for the non specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.006. Non Specific Colour % (NSCliving %) was calculated as 0.8 %. This is below the threshold of 5 %, therefore correction due to colouring potential was not necessary.

VIABILITY RESULTS
The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 1. The mean OD value for the test material treated skin samples showed a 99.3 % relative viability.

ACCEPTANCE OF RESULTS:
-After receipt, the two indicators of the delivered kit were checked in each case. Based on the observed colours, the epidermis units were in proper conditions.
-The mean OD value of the two negative control tissues was in the recommended range (0.836).
-The two positive control treated tissues showed 0.8 % viability demonstrating the proper performance of the assay.
-The difference of viability between the two test material-treated tissue samples in the MTT assay was 5.4 %.
-The difference of viability between the two negative control tissue samples in the MTT assay was 15.0 %.
-The mean OD value of the blank samples (acidified isopropanol) was 0.046.
All these parameters were within acceptable limits and therefore the study was considered to be valid.

Any other information on results incl. tables

Table 1: Optical Density (OD) and the calculated relative viability % of the samples

Substance	Optical density (OD)			Viability (% RV)
		Measured	Blank corrected
Negative control	1	0.819	0.773	92.5
	2	0.944	0.898	107.5
	Mean	-	0.836	100.0
Positive control	1	0.052	0.006	0.8
	2	0.053	0.007	0.8
	Mean	-	0.007	0.8
Test material	1	0.853	0.807	96.6
	2	0.898	0.852	101.9
	Mean	-	0.830	99.3

Mean blank value was 0.046.

Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

Applicant's summary and conclusion

Interpretation of results:

other: Not corrosive in accordance with EU criteria

Conclusions:

Under the conditions of the study, the test material is non-corrosive to the skin.

Executive summary:

The potential of the test material to cause corrosion to the skin was determined in accordance with the standardised guidelines OECD 431 and EU Method B40.Bis using the reconstructed human epidermis model, under GLP conditions.

EPISKIN™ (SM) is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.

Disks of EPISKIN™ (SM) (two units) were treated with the test material and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9 % (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving %) from the test material. For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35 % of the negative control, the test material is considered to be corrosive to skin.

Following exposure with the test material, the mean cell viability was 99.3 % compared to the negative control. This is above the threshold of 35 %, therefore the test material was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

Under the conditions of the study, the test material is non-corrosive to the skin.