Fatty acids, tall-oil, reaction products with linseed oil, maleic anhydride and methanol

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Addition product of maleic anhydride, tall oil fatty acids, linseed oil and methanol

IUPAC Name:

Addition product of maleic anhydride, tall oil fatty acids, linseed oil and methanol

Test material form:

liquid

Details on test material:

UVCB, Purity 100%, brown liquid, homogenous, Batch no.210150084, Expiry date 18 May 2016

Specific details on test material used for the study:

Being the test item a viscous/not dispensable substance, a preliminary trial was performed in order to verify the best way to carry out the treatment. The following trials were performed:
– An aliquot of the test item was frozen in order to be reduced into powder. No relevant change in test item physical state was noted after 3 days at -80 °C.
– Two aliquots of the test item were warmed for approximately 1 hour. No relevant change in test item texture was observed after warming at 37 or 45 °C.
– An aliquot of test item was withdrawn with a 1 mL syringe and 25 mg was weighed directly on the surface of the plate. A sufficiently accurate weight was obtained. Moreover, the test item could be mechanically removed from the well with a cotton stick.

Based on these results, in the preliminary trial the test item was weighed directly on the surface of the tube (Colouring potential test) or plate (Direct MTT reduction test), while in the Main Assay the test item was weighed directly on the surface of each tissue.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other:

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin, SkinEthic Laboratories, Lyon, France
- Tissue batch number(s):15-EKIN-040
- Production date:
- Shipping date:
- Delivery date:
- Date of arrival at laboratory: 6 Oct 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 deg C
- Temperature of post-treatment incubation (if applicable):

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1
- Observable damage in the tissue due to washing:
- Modifications to validated SOP:


FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues
- Procedure used to prepare the killed tissues (if applicable):
- N. of replicates :
- Method of calculation used:

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439:SKIN DISC PREPARATION
- Procedure used: Not different; cut-off point cell viability /= 18
- Quality control for skin discs:
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 +/- 2 deg C
- Temperature of post-treatment incubation (if applicable): same
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 1
- Observable damage in the tissue due to washing:
- Modifications to validated SOP:
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT [3-(4,5-Dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS RN. 298-93-1]
- Spectrophotometer:
- Wavelength:
- Filter:
- Filter bandwidth:

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

20.2 mg/epidermal unit

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Test animals

Details on test animals or test system and environmental conditions:

A commercial reconstructed human skin product, EPISkin, was used in the study.

Test system

Preparation of test site:

not specified

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

no

Amount / concentration applied:

20 mg

Duration of treatment / exposure:

15 minutes

Observation period:

43 hours

Details on study design:

Maintenance Medium SkinEthic; batch: 15-MAIN3-041
Assay Medium SkinEthic; batch: 15-ESSC-041

Preliminary test: Direct MTT reduction test (Step 1):

Non-specific reduction of MTT was evaluated as follows: two mL of MTT Ready-to-use Solution was incubated with 21.25 mg of test item at 37°C, 5% CO2 and saturated humidity for 3 hours protected from light, simulating test conditions. Observation of blue or purple appearance of the solution at the end of the incubation time was carried out.

Colouring potential test (Step 2)
Chemicals’ colouring potential was assessed for potential interaction with the test system. 11.67 mg of the test item was added to 90 µL of distilled water in a transparent tube and the resulting solution/suspension mixed by using a vortex for 15 minutes. Colouring of the solution/suspension at the end of the incubation time was evaluated.

Main test:
A Main Assay was carried out including the test item, positive and negative controls. Results presented in this report were obtained in a repeated assay. In the original experiment the negative control showed a mean Optical Density value (OD = 0.523) out of the acceptability range (OD >/= 0.600 and At the end of the 15 minute exposure, each tissue was rinsed with approximately 25 mL of sterile D-PBS filling and emptying the tissue insert of the plate. The test item was mechanically removed with cotton sticks and then the remaining substance was washed with sterile D-PBS, filling and empting the tissue insert, until reaching the complete removal of the test item. The excess liquid was carefully removed and the sample transferred in new wells pre-filled with 2 mL/well of maintenance medium.

Post-exposure period
A 43 hour recovery period was allowed by incubation at 37°C, 5% CO2 and saturated humidity.

MTT Assay
Each tissue insert was incubated with 2 mL/well of MTT ready-to-use solution for approximately 3 hours at 37°C, 5% CO2 and saturated humidity. At the end of the incubation period, tissues were placed on absorbent paper to dry. A total biopsy was carried out by means of a biopsy punch to allow biopsies of the same dimensions.
The epidermis was separated from the collagen matrix and both placed in a microtube prefilled with 500 µL of acidic isopropanol. Tubes were preserved for approximately 3 days at 4°C to allow formazan extraction. At the end of the extraction period, debris were eliminated by short centrifugation of the tubes (approximately 11000 rpm for 2 minutes) and aliquots of 200 µL from each sample were read in duplicate for their absorbance at 595 nm. OD values were recorded. Six aliquots (200 µL) of acidic isopropanol were analysed and used as blank.

Sample Test System Treatment Amount per well Number of replicates Sample code
Negative Live D-PBS 20 µL 3 N1 N2 N3
control tissue
Positive Live 5% SDS in water 20 µL 3 P1 P2 P3
control tissue
Test item Live
tissue ZWA 5496/100 20± 2 mg 3 A1 A2 A3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

main study

Value:

36

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Of 3 replicates, two showed viability > 40% (44.6 and 45.0%, respectively), while one showed viability of 18.5%. The average was 36.0% viability with a SD viability of 15.2.

Any other information on results incl. tables

PRELIMINARY TEST

 

 

 

Direct MTT reduction test (Step 1)

 

Test item (mg)	MTT ready to use solution(mL)	Container	Incubation condition	Colour Observation
20	2.0	well	3 h at 37°C, 100% nominal humidity 5% CO2	No colour change (no interaction)

 

 

Colouring potential test (Step 2)

 

Test item (mg)	Water (mL)	Container	Incubation condition	Colour Observation
10	90	Eppendorf tube	15’, ambient condition, in agitation	No colour change (no interaction)

MAIN ASSAY

 

BLANK                   NegativeControl

 

OD		OD	OD-blank                                                              Viability(%)
0.079	N1-1	0.821	0.7392                           0.7647                                     113.5
0.081	N1-2	0.872	0.7902
0.082	N2-1	0.698	0.6162                           0.6342                                     94.1
0.083	N2-2	0.734	0.6522
0.083	N3-1	0.675	0.5932                           0.6222                                     92.4
0.083	N3-2	0.733	0.6512

Mean        0.0818                   Mean        0.7555                              Mean       0.67370     -------> 100.0

SD        0.0016                      SD        0.0757                                 SD      0.07904                                   11.7

CV(%)           1.96                CV(%)          10.02                           CV(%)          11.73                                 11.70

Positive Control

0.159 0.171 0.111 0.232 0.102 0.108

P1-1 P1-2 P2-1 P2-2 P3-1 P3-2

 

 

OD   OD-blank                                                                Viability(%)

0.0772                           0.0832                                       12.3

0.0892

0.0292                           0.0897                                       13.3

0.1502

0.0202                           0.0232                                         3.4

0.0262

Mean        0.1472                              Mean       0.06537     -------> 9.7

SD        0.0506                                 SD      0.03666                                     5.5

CV(%)          34.38                           CV(%)          56.08                                 56.70

 

 

Test Item	OD	OD-blank                                                                 Viability(%)
A1-1	0.183	0.1012                           0.1247                                     18.5
A1-2	0.230	0.1482
A2-1	0.374	0.2922                           0.3007                                     44.6
A2-2	0.391	0.3092
A3-1	0.379	0.2972                           0.3032                                     45.0
A3-2	0.391	0.3092
Mean	0.3247	Mean	0.24287	------->	36.0
SD	0.0930	SD	0.10234		15.2
CV(%)	28.64	CV(%)	42.14		42.22

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The EPISKIN in vitro model was found to be positive, (considered an irritant), based on the mean cell viability (36.0%) when compared to the negative control. the test em is considered irritant.