Fatty acids, linseed-oil, reaction products with 2-amino-2-(hydroxymethyl)-1,3-propanediol and formaldehyde

Endpoint summary

Description of key information

The potential of VOELOFA Monomer (target substance) to induce eye irritation was tested in a suitable in vitro test method (OECD 492). In accordance with REACH regulation 1907/2006, column 2 of standard information requirement 8.1 it is not necessary to conduct an in vitro skin irritation/corrosion study as in an acute toxicity study by the dermal route no indication of skin irritation was noted at the limit dose of 2000 mg/kg bw. Based on the results, the target substance can be considered non-irritant to the skin and eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because an acute toxicity study by the dermal route does not indicate skin irritation up to the relevant limit dose level (2 000 mg/kg body weight)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

2016-07-06 to 2016-09-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method: The EpiOcular^TM Eye Irritation Test (EIT) predicts the acute ocular irritation potential of chemicals by measurement of its irreversible tissue damage caused by cytotoxic effects in the human cornea model. Within a testing strategy, the EpiOcularTM EIT is used as a re-placement of the Draize Eye Irritation Test.

- Description of the cell system used: The EpiOcularTM tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcular^TM tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm²

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL

Duration of treatment / exposure:

28 minutes at 37 °C

Observation period (in vivo):

n.a.

Duration of post- treatment incubation (in vitro):

Post exposure post-soak plate: 12 min at room temperature
Post exposure post-treatment plate: 116 ± 4 min at 37 ± 1 °C

Number of animals or in vitro replicates:

2 tissues per dose group

Details on study design:

- Details of the test procedure used:
On the day of the start of the experiment, the MTT concentrate was thawed. The concentrate was diluted with the MTT solvent and the solution was stored at 2 - 8 °C in the dark. The assay medium was warmed in the water bath to 37 ± 1 °C. 6-well-plates were labelled with test item, resp. negative control, resp. positive control and filled with 1 mL assay medium in the appropriate wells. All 24 inserts were inspected for viability and the presence of air bubbles between agarose gel and insert. Viable tissues were transferred in the prepared 6-well-plate and incubated at 37 ± 1 °C, 5 ± 1% CO2 and 80 – 100 % relative humidity for 1 hour. After the pre-incubation, the medium was replaced and the wells were filled with 1 mL fresh assay medium. All 6-well-plates were incubated at 37 ± 1 °C, 5 ± 1% CO2 and 80 – 100% relative humidity for 16.5 hours (16 – 24 hours).
After overnight incubation, the tissues were pre-wetted with 20 µL DPBS buffer and then incubated at 37 ± 1 °C, 5 ± 1% CO2 and 80 – 100 % relative humidity for 30 minutes. Af-ter that, 50 µL of the controls and the test item were applied in duplicate in one-minute-intervals. This was done in such a fashion that the upper surface of the tissue was cov-ered. At the beginning of each experiment (application of negative controls), a stop watch was started. After dosing the last tissue of each plate, the plate was transferred into the incubator for 28 minutes at 37 ± 1 °C, 5 ± 1% CO2 and 80 – 100 % relative humidity. At the end of the exposure time, the inserts were removed from the plates in one-minute-intervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately transferred to and immersed in 5 mL of pre-warmed assay medium in a pre-labelled 12-well plate for 12 minutes post soak at room temperature.
After that, each insert was removed from the medium, the medium was decanted off the tissue and the insert was blotted on absorbent material and transferred into the respective well of a pre-labelled 6-well plate containing 1 mL assay medium. For post-treatment incubation, the tissues were incubated for 116 ± 4 minutes at 37 ± 1 °C, 5 ± 1% CO2 and 80 – 100% relative humidity. After the post-treatment incubation, the MTT Assay was performed. Thus, a 24-well-plate was prepared with 300 µL freshly prepared MTT-reagent in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solution. The plate was incubated for 180 minutes at 37 ± 1 °C, 5 ± 1% CO2 and 80 – 100% relative humidity.
At last, each insert was thoroughly dried and set into the empty 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was firmly sealed to avoid evaporation of the solvent and stored in the refrigerator overnight. On the next day the plate was shaken for 2 hours at room temperature. The inserts were pierced with an injection needle, taking care that all colour is extracted. The inserts were then discarded and the content of each well was thoroughly mixed in or-der to achieve homogenisation. From each well, two replicates with 200 µL solution (each) were pipetted into a 96-well-plate. Eight wells with 200 µL isopropanol were pipetted, too. The plate was read in a plate spectral photometer at 570 nm.

- RhCE tissue construct used, including batch number:
The EpiOcular^TM tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcular^TM tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells.
The EpiOcular™ tissues were provided as kit (e.g. OCL-200-EIT, batch 23719; MatTek).

- Doses of test chemical and control substances used:
1. Negative Control: 50 µL sterile water (Laus GmbH, batch 20160426)
2. Positive Control: 50 µL methyl acetate (CAS No. 79-20-9 (positive control; Lot No.: 102015ZSA)
3. Test Item: 50 µL

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable):
Exposure: 28 min at 37 ± 1 °C, 5% CO2 and 80-100% relative humidity
Post exposure post-soak plate:12min at room temperature
Post exposure post-treatment plate: 116 ± 4 min at 37 ± 1 °C, 5% CO2 and 80-100% relative humidity

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals:
See section "Pre-experiments" in box "Any other information on materials and methods incl. tables"

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): 2 tissues per group

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
Mean tissue viability (% negative control) <= 60 %: GHS “Category 1” or “Category 2”
Mean tissue viability (% negative control) > 60%: No GHS Category for eye irritation

Test Acceptance Criteria:
- mean absolute OD570 nm of the negative control is ≥ 0.8 and ≤ 2.5
- mean relative tissue viability of the positve control is < 50% of negative control
- Variation within replicates < 20%

Irritation parameter:

other: Relative Tissue Viability (%)

Run / experiment:

Mean of replicates

Value:

97.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was < 60% (97.2%). For detailed information please refer Tables 1-6 in box "Any other information on results incl. tables" .

Table 1: Absorbance Values Blank Isopropanol (OD at 570 nm)

Replicate	1	2	3	4	5	6	7	8	Mean
Absorbance	0.037	0.037	0.034	0.036	0.036	0.037	0.036	0.037	0.036

 

Table 2: Absorbance Values Negative Control, Positive Control and Test Item (OD at 570 nm)

Designation	Measurement	Negative Control	Positive Control	VOELOFA Monomer
Tissue 1	1	2.043	0.548	1.916
	2	1.957	0.530	1.865
Tissue 2	1	1.936	0.555	1.907
	2	1.901	0.529	1.929

 

Table 3: Mean Absorbance Negative Control, Positive Control and Test Item

Designation	Negative Control	Positive Control	VOELOFA Monomer
Mean – blank (Tissue 1)	1.964	0.503	1.855
Mean – blank (Tissue 2)	1.883	0.506	1.882

 

Table 4: % Viability Positive Control and Test Item

Designation	Positive Control	VOELOFA Monomer
% Viability (Tissue 1)	26.2%	96.4%
% Viability (Tissue 2)	26.3%	97.9%
% Viability Mean	26.2%	97.2%

 

Table 5: Assessment of Eye Irritation

% Viability	Assessment	GHS classification
> 60 %	Non eye irritant	No GHS category for eye irritation
≤ 60 %	Eye irritant	GHS category 1 or 2

 

Table 6: Validity

Criterion	Demanded	Found
OD of negative control	≥ 0.8 and ≤ 2.5	1.9
% Formazan production of positive control	< 50% of negative control	26.2%
Variation within replicates	< 20%	4.2% (negative control) 0.2% (positive control) 1.4% (test item)

 

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no irritant effects. The test item is classified as “non-irritant“ in accordance with UN GHS “No Category” for eye irritation.

Executive summary:

In the present study the eye irritant potential of VOELOFA Monomer (100% purity) was analysed according to OECD 492 using the three-dimensional human corneal epithelium model EpiOcular, consisting of normal, human-derived epidermal keratinocytes mimicking characteristics of the corneal epithelium. Hereby, 50 µL of the test item was applied directly atop the EpiOcular™ tissue. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT compared to those of the concurrent negative control. The test item showed no irritant effects. The mean relative tissue viability of two replicates (% negative control) was > 60% (97.2%). Therefore, the test item is considered to be non-irritating to the eye in accordance with UN GHS “No Category”.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The potential of VOELOFA Monomer (target substance) to induce eye irritation was tested in a suitable in vitro test method (OECD 492). In accordance with REACH regulation 1907/2006, column 2 of standard information requirement 8.1 it is not necessary to conduct an in vitro skin irritation/corrosion study as in an acute toxicity study by the dermal route no indication of skin irritation was noted at the limit dose of 2000 mg/kg bw. Based on the results, the target substance can be considered non-irritant to the skin and eye.

Justification for classification or non-classification

Based on the available results, the target substance can be considered non-irritant to the skin and eye and thus, no classification is warranted.