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

Endpoint summary

Description of key information

The substance is a skin irritant in an in vitro rHSE model (EPISKIN) and is a non-irritant in an ex vivo BCOP model.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

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– An aliquot of test item was withdrawn with a 1 mL syringe and 25 mg was weighed directly on the surface of the plate.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.

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

Details on test animals or test system and environmental conditions:

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

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

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.

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

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

yes

Remarks:

minor. The test item was stored at 25.9 deg C for 8.5 h, rather than at 20 deg C as stated in the protocol. Also,the measurement of opacity was done in a photometer rather than an opacitometer, but an appropriate calculation can be made for correction.

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

GLP compliance:

yes

Species:

cattle

Strain:

other: Bos primagenius Taurus

Details on test animals or tissues and environmental conditions:

Bovine corneas were used from previously slaughtered cattle which were between ages 12-60 months. SOURCE OF COLLECTED EYES
- Source:
- Number of animals:
- Characteristics of donor animals (e.g. age, sex, weight): age 12-60 months, M/F,
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
- Time interval prior to initiating testing:
- indication of any existing defects or lesions in ocular tissue samples: eyes checked for defects as part of the selection criteria
- Indication of any antibiotics used:
TEST ANIMALS
- Source: Muller Fleisch GmbH,Birkenfeld, Germany. Isolated fresh.
- Age at study initiation:12-60 months
- Weight at study initiation: No data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): After receipt, incubated 1 h at 32 deg C in Hank's Balanced Salt Solution in exposure chambers. Vessels were sterilized glass or sterilizable plastic.
- Humidity (%):
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):

IN-LIFE DATES: 2 Sept. 2015

Vehicle:

other: Minimal Essential Medium (MEM) without penol but supplemented with sodium bicarbinate, L-glutamine and 10% fetal calf serum (FCS).

Controls:

yes, concurrent positive control

yes, concurrent negative control

no

Amount / concentration applied:

750 μL

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

Bovine eyes ex vivo were used in the study.

Details on study design:

Light transmission was recorded through each cornea in a spectrophotomer at 570 nm, prior to exposure. This comprised the baseline opacity.

Three replicate measurements were obtained.

The closed chamber method was used. The respective substance (negative control solution, test item or positive control) was applied by pipetting 750 μL of the appropriate liquid through the refill hole in the holder of the cornea. The test item was applied to the epithelium in such a manner that as much as possible of the cornea was covered with the test item.

Exposure time was 10 min at 32 ± 1 °C. After rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, the anterior chamber was filled with cMEM without phenol red, and the corneas were stored for additional 2 h at 32 ± 1 °C (post-incubation). The cMEM without phenol red was renewed in both chambers of the apparatus. Then, the final opacity value of each cornea was recorded (again by measurement at 570 nm).

The cMEM without phenol red was removed from the front chamber, and 1 mL sodium fluorescein solution (concentration: 4 mg/mL) was added to the front chamber. The chambers were then closed and incubated for 90 min at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, the permeability of the cornea was measured by reading the optical density at 490 nm of the liquid in the posterior chamber.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

1.83

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Mean opacity difference of the negative control is 0.1220.
For the test item, the calculated IVIS (In vitro irritancy scoe) is 1.83. The experiment is considered as sufficient for the classification of the test item because two of the three replicates of the test item lead to the same assessment for the test item.

Optical Density of the test substance:

Replicate	Negative Control			Test Item ZWA 5496/100			Positive Control
Measured values	-0.0004	0.0007	0.0031	0.0060	0.0413	0.0240	0.1997	0.2629	0.2637
*Corrected values	-0.0020	0.0035	0.0155	0.0300	0.2065	0.1200	0.9985	1.3145	1.3185
Mean	0.0057

Absorbance and Opacity Values of the Negative Control:

Parameter	Negative Control
Absorbance before exposition	0.1888	0.1907	0.1792
Absorbance after exposition	0.2395	0.2033	0.2148
Opacity before exposition	1.5445	1.5513	1.5108
Opacity after exposition	1.7358	1.5970	1.6398
Opacity Difference	0.1913	0.0457	0.1291

Absorbance and Opacity Values for the Test Item and the Positive Control:

Parameter	Test Item ZWA 5496/100			Positive Control
Absorbance before exposition	0.1297	0.2813	0.1462	0.1425	0.1296	0.1766
Absorbance af- ter exposition	0.1560	0.3495	0.2432	1.9991	1.8398	1.9957
Opacity before exposition	1.3480	1.9112	1.4002	1.3884	1.3477	1.5018
Opacity after exposition	1.4322	2.2361	1.7507	99.7930	69.1512	99.0148
Opacity Difference	0.0842	0.3250	0.3504	98.4046	67.8035	97.5130

In Vitro Irritancy Scores:

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative Control 0.9% NaCl	0.16	0.21	66.4%
	0.10
	0.36
Test Item ZWA5496/100	0.33	1.83	79.2%
	3.22
	1.94
Positive Control DMF undiluted	113.18	105.86	15.3%
	87.31
	117.08

Note: the high relative standard deviations of the IVIS of test item and negative control are due to mathematical reasons, as the respective means are very small.

Interpretation of results:

GHS criteria not met

Conclusions:

The calculated IVIS (in vitro irritancy score) of the undiluted test substance in the BCOP is 1.83. The criteria for a non-irritant (IVIS of 3 or less) is met and the substance is evaluated as not irritating to the eye.

Executive summary:

less

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In the OECD 439 in vitro skin irritation test, two of three values for cell viability suggested that the substance is not a skin irritant, but the mean value of three replicates resulted in a value below the threshold of 40% viability. Additional study of the test material in other models may be indicated. However, since the material is found to be a dermal sensitiser in the LLNA, efforts should be made to minimise dermal exposure of workers.

In the OECD 437 BCOP ex vivo model of eye irritation, the substance is found to be non-irritating, with an irritancy score below 3 (threshold for non-irritant).

An IATA for skin irritation was undertaken with conclusions from a Weight-of-Evidence analysis (Element 7) that the substance is not a skin corrosive. This includes an absence of physico-chemical alerts, available information from an in vivo skin sensitisation study in mice (LLNA) indicating irritation, in vitro data indicating irritation, and absence of reports of adverse skin reactions among human workers.

Justification for classification or non-classification

The substance was found to be a skin irritant in an in vitro (EPISKIN) model. This data, combined with data from a LLNA, indicate classification of the substance as a skin irritant, Category 2, according to Regulation EC No. 1272/2008.

The substance is not an eye irritant, according to the BCOP ex vivo assay, and does not meet the criteria for classification for eye damage or irritation.