Fatty acids, tall-oil, polymers with glycidyl neodecanoate

Endpoint summary

Description of key information

The test substance is not irritating to skin and eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 22, 2015 to June 26, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

Human three dimensional epidermal model (EpiDerm (EPI-200)). Obtained from MatTek In Vitro Life Science Laboratories, Bratislava.

Justification for test system used:

Guideline recommended.

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent no treatment

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

30 μL

Duration of treatment / exposure:

60 min

Details on study design:

PERFORMANCE OF THE STUDY:

Additional tests:
1. Nylon mesh compatibility: The test substance was tested for possible reaction with the nylon mesh which is used to ensure sufficient contact with the tissue surface. 30 μL test substance were brought onto a nylon mesh on a microscope slide. No reaction with the mesh was visible after 1 h exposure.
2. Assessment of coloured or staining test substance: It was tested whether the test substance develops a colour without MTT addition. 30 μL test substance were given in a test tube with 0.3 mL H2O demineralised and incubated at 37±1°C and 5±0.5% CO2 for 60 min. The resulting solution was colourless, therefore no binding capacity had to be tested.
3. Assessment of direct reduction of MTT by the test substance: The test substance was tested for the ability of direct MTT reduction. To test for this ability, 30 μL test substance were added to 1 mL of MTT solution and the mixture was incubated in the dark at 37±1°C and 5±0.5% CO2 for 60 min. Untreated MTT solution was used as control. The MTT solution did not change its colour within 1 h, therefore, direct MTT reduction had not taken place, and no data correction was necessary.

Pre-Incubation of tissues:
Eight 6-well-plates were prepared with 0.9 mL assay medium in 3 of the 6 wells (upper row). The tissues were inspected for viability. Viable tissues were transferred (3 per plate) in the wells with the medium using sterile forceps under the clean bench and placed into the incubator at 37±1°C and 5±0.5% CO2 for 1 h. After the pre-incubation (1 h), the other 3 wells of each plate (lower row) were filled with fresh assay medium (0.9 mL). Every tissue was transferred into a well of the lower row. All 6-well-plates were set into the incubator at 37±1°C and 5±0.5% CO2 for 18 h.

Treatment:
The pre-incubated tissues were placed into fresh 6-well-plates containing 0.9 mL assay medium per well, using the upper row only. One plate (three tissues) was used as negative control; each tissue was treated with 30 μL DPBS buffer, a nylon mesh was added in order to ensure sufficient contact with the tissue surface. One plate was used as positive control; each tissue was treated with 30 μL SDS-solution, a nylon mesh was added in order to ensure sufficient contact with the tissue surface. One plate was used for treatment with the test substance: 30 μL test substance was applied, and a nylon mesh was added in order to ensure sufficient contact with the tissue surface. Tissues were dosed in 1 min. intervals. After dosing the last tissue, all plates are transferred into the incubator for 35 min. 37±1°C and 5±0.5% CO2 60 min. after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-min.-intervals. After rinsing, each tissue was dried with a sterile cotton tip and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL). Then, the tissues were set in the incubator for 24 h.

Medium renewal:
For three incubated tissues, a new 6-well-plate with 0.9 mL assay medium in the upper row was prepared. The tissues were removed from the incubator and shaken for 10 min (500 rpm). Then the inserts were transferred into the new 6-well-plate and set into the incubator for 18 h for post-incubation.

MTT assay:
After a total incubation time of 42 h, a 24-well-plate was prepared with 300 μL freshly prepared MTT-reagent in each well. The tissues were blotted on the bottom and then transferred into the 24-well-plate. Then the 24-well-plate was set into the incubator for 3 h. After this time, the MTT reagent was aspirated and replaced by DPBS buffer. This was then aspirated, too, and replaced several times. At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was then shaken for 2 h at room temperature. After 2 h, the inserts in which formazan had been produced were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homogenisation. From each well, two replicates with 200 μL solution (each) were pipetted into a 96-well plate which was read in a plate spectral photometer at 570 nm.

Irritation / corrosion parameter:

other: other: relative absorbance (%)

Value:

97.8

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

no indication of irritation

- Comparison of formazan production:

For the test substance and the positive control, the following percentage values of formazan production were calculated in comparison to the negative control:

Table: Formazan production

Designation	Test substance	Positive control
% Formazan production (tissue 1)	93.7%	4.5%
% Formazan production (tissue 2)	95.7%	3.7%
% Formazan production (tissue 3)	103.9%	4.5%
% Formazan production (mean)	97.8%	4.2%

 

The relative absorbance values were reduced to 97.8% after the treatment. This value is above the threshold for skin irritation (50%). Therefore, the test substance was considered as not irritant to skin.

 

- Validity and acceptability:

All validity criteria were met. Values for negative control and for positive control were within the range of historical data of the test facility. Therefore, the experiment was considered valid.

Interpretation of results:

not irritating

Conclusions:

The test substance is non irritating to skin.

Executive summary:

A study was conducted to evaluate the skin irritation potential of the test substance in an in vitro human skin model according to OECD Guideline 439 and EU Method B.46, in compliance with GLP. The test consisted of a topical exposure to neat test substance (30 µl) of a human reconstructed epidermis model followed by a cell viability assay. After treatment, the relative absorbance values were reduced to 97.8%. This value was well above the threshold for skin irritation (50%). The optical density (OD) of the negative control was well within the required acceptability criteria. The positive control induced a decrease in the relative absorbance as compared to the negative control down to 4.2%. Variation within replicates was within the accepted range for negative and positive controls, and test substance. Under the study conditions, the test substance was found to be not irritating to skin (Andres, 2015).

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:

From June 29, 2015 to July 8, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the testing of chemicals, series on testing and assessment no. 160: “Guidance document on “the bovine corneal opacity and permeability (bcop) and isolated chicken eye (ice) test methods.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: Bos primigenius taurus (fresh bovine corneas)

Details on test animals or tissues and environmental conditions:

- Species: Bos primigenius Taurus (fresh bovine corneas).
- Source: Slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany.
- Age of cattles: Between 12 and 60 months old.
- Transport of eyes: In Hank’s balanced salt solution (supplemented with 0.01% streptomycin and 0.01% penicillin).

Vehicle:

unchanged (no vehicle)

Controls:

other: Negative control: 0.9% NaCl; positive control: Dimethylformamide, DMF

Amount / concentration applied:

TEST SUBSTANCE
- Amount applied: 750 μL

Duration of treatment / exposure:

10 min

Observation period (in vivo):

2 h

Number of animals or in vitro replicates:

3 replicates

Details on study design:

PREPARATION OF TEST MATERIAL
The test substance was tested directly, without dilution or preparation of a solution.

NEGATIVE CONTROL
Sodium chloride solution: 0.9% NaCl (CAS No. 7647-14-5), dissolved in demineralised water.

POSITIVE CONTROL
Dimethylformamide, DMF, CAS no. 68-12-2, undiluted.

PREPARATIONS
After having carefully cleaned and sterilized the cornea holders, they were kept in the incubation chamber at 32±1°C. On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (complete MEM) and stored in a water bath at 32±1°C. The same was performed with the MEM with phenol red but without addition of sodium bicarbonate. After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2 - 3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM without phenol red was filled. The holders were then incubated for 1 h in the incubation chamber at 32±1°C.

METHOD
After the initial incubation of 1 h, the medium was changed and the baseline opacity for each cornea was recorded at 570 nm. For each treatment group (negative control solution, test substance and positive control), three replicates were used. After removal of the pre-incubation medium, 750 μL were applied to each replicate by closed chamber-method. An exposure period of 10 min was given. After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, corneas were stored for additional 2 h at 32±1°C. Then, the final opacity value and permeability of each cornea was recorded.

Irritation parameter:

in vitro irritation score

Remarks:

mean IVIS

Value:

0.62

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

The test substance showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) was 0.62 under the conditions of this test.

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

Table 1: Calculated and mean IVIS

Test group	IVIS	Mean IVIS	Relative standard deviation IVIS
Negative Control 0.9% NaCl	-0.88	-0.75	-14.8%
	-0.70
	-0.67
Test substance	1.08	0.62	91.7%
	0.80
	-0.02
Positive control DMF undiluted	94.18	105.62	35.3%
	75.43
	147.25

 

The test was considered as valid if the positive control causes an IVIS that falls within two standard deviations of the current historical mean. The negative control has to show an IVIS ≤3. Values for negative and positive controls were within the range of historical data of the test facility. Therefore, the test system was acceptable.

Interpretation of results:

not irritating

Conclusions:

The test substance is not irritating to eyes.

Executive summary:

An in vitro study was conducted to assess the corneal damage potential of the test substance by quantitative measurements of changes in opacity and permeability in a bovine cornea, according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. The test substance was brought onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32±1°C for 1 h and whose opacity had been determined. The test substance was incubated on the cornea for 10 min at 32±1°C. After removal of the test substance and 2 h post incubation, opacity and permeability values were measured and compared with the initial value. The negative and positive controls met the validity criteria. The test substance showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) was 0.62. Under the test conditions, the test substance was found to be not irritating to eyes (Andres, 2015).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro skin irritation test

A study was conducted to evaluate the skin irritation potential of the test substance in an in vitro human skin model according to OECD Guideline 439 and EU Method B.46, in compliance with GLP. The test consisted of a topical exposure to neat test substance (30 µl) of a human reconstructed epidermis model followed by a cell viability assay. After treatment, the relative absorbance values were reduced to 97.8%. This value was well above the threshold for skin irritation (50%). The optical density (OD) of the negative control was well within the required acceptability criteria. The positive control induced a decrease in the relative absorbance as compared to the negative control down to 4.2%. Variation within replicates was within the accepted range for negative and positive controls, and test substance. Under the study conditions, the test substance was found to be not irritating to skin (Andres, 2015).

 

Bovine corneal opacity and permeability test

An in vitro study was conducted to assess the corneal damage potential of the test substance by quantitative measurements of changes in opacity and permeability in a bovine cornea, according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. The test substance was brought onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32±1°C for 1 h and whose opacity had been determined. The test substance was incubated on the cornea for 10 min at 32±1°C. After removal of the test substance and 2 h post incubation, opacity and permeability values were measured and compared with the initial value. The negative and positive controls met the validity criteria. The test substance showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) was 0.62. Under the test conditions, the test substance was found to be not irritating to eyes (Andres, 2015).

Justification for selection of skin irritation / corrosion endpoint:
The study was conducted according to OECD Guideline 439 and EU Method B.46, in compliance with GLP.

Justification for selection of eye irritation endpoint:
The study was conducted according to OECD Guideline 437 and EU method B.47, in compliance with GLP.

Justification for classification or non-classification

Skin irritation

Based on the results of an in vitro skin irritation study, the test substance does not need to be classified for this endpoint according to the EU CLP criteria (EC 1272/2008).

Eye irritation

Based on the results of a bovine corneal opacity and permeability test, the test substance does not need to be classified for this endpoint according to the EU CLP criteria (EC 1272/2008).