Fatty acids, tall-oil, reaction products with triethylenetetramine

Endpoint summary

Description of key information

The test substance was found to be non irritating to the skin, while it was irritating to the eyes.

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

Study period:

From February 24, 2015 to March 06, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD Guideline 439 and EU Method B.46, in compliance with GLP.

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)

Species:

other: EpiDerm-Kit consisting of human-derived epidermal keratinocytes, Batch no.: 21630

Type of coverage:

open

Vehicle:

other: Dulbecco’s Phosphate Buffered Saline (DPBS buffer without CaCl2 and MgCl2)

Controls:

other: in vitro study: Dulbecco’s Phosphate Buffered Saline (DPBS buffer without CaCl2 and MgCl2) was used as negative control. Sodium dodecyl sulphate (SDS) solution in deionised H2O containing 5% SDS was used as positive control.

Amount / concentration applied:

24.1, 24.2, 24.4 mg test substance (Main test); 26.3, 26.9 mg test substance (Additional test with frozen tissues).
On average, 25 mg of the solid test substance (wetted with 25 μL DPBS-buffer) were applied to each tissue and spread to match the tissue size of 0.63 cm2

Duration of treatment / exposure:

60 minutes

Details on study design:

Test System: The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell cultures inserts. Tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava.

Pre-Incubation of Tissues:
Eight 6-well-plates were prepared with 0.9 mL assay medium in three of the six wells (upper row). The tissues were inspected for viability. Viable tissues were transferred (three 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±0.5% CO2 for 1 h.

After the pre-incubation (1 h), the other three 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±0.5% CO2 for 18 h.

Treatment with test substance:
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:
The tissues were wetted with 25 μL DPBS buffer before applying the test substance and spreading it to match the tissue size.
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 each, 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 ten 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-wellplate which was read in a plate spectral photometer at 570 nm.

Irritation / corrosion parameter:

other: other: Relative absorbance value (percent)

Value:

97.9

Remarks on result:

other:

Remarks:

Basis: mean (in comparison to the negative control). Time point: 60 min. Remarks: Value was well above the threshold value for irritation potential (50%) . (migrated information)

Irritant / corrosive response data:

After the treatment, the relative absorbance values were reduced to 97.9%. This value is well above the threshold value for skin irritation (50%).
The optical density of the negative control was well within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8 with an OD value of 2.3. The positive control induced a decrease in the relative absorbance as compared to the negative control to 2.8% (required: ≤20%) for thus ensuring the validity of the test system. Variation within replicates was within the accepted range for negative control, positive control and test substance (< 18%). For these reasons, the result of the test is considered valid.

Table 1: Corrected Mean Absorption Values of Test Substance

Designation	Test substance
Mean – blank (tissue 1)	2.256
Mean – blank (tissue 2)	2.121
Mean – blank (tissue 3)	2.230
Mean of the three tissues	2.202
Relative standard deviation of the three tissues	3.3%

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 2: Percent Formazan Production

Designation	Test substance	Positive control
Percent formazan production (tissue 1)	100.3%	2.8%
Percent formazan production (tissue 2)	94.3%	2.8%
Percent formazan production (tissue 3)	99.1%	2.9%
Percent formazan production (mean)	97.9%	2.8%

Conclusions:

The test substance was considered as not irritating to the skin.

Executive summary:

An in vitro study was conducted to assess the skin irritation potential of the test substance according to OECD Guideline 439 and EU Method B.46, in compliance with GLP. Three tissues of a human skin model EpiDermTM were exposed for 60 minutes. On average, 25 mg of the test substance (wetted with 25 μL DPBS-buffer) were applied to each tissue and spread to match the tissue size (0.63 cm²). DPBS buffer was used as negative control and 5% SDS solution as positive control. After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, with an OD value of 2.3. The positive control showed clear irritating effects with a relative absorbance value reduced to 2.8%. Variation within tissues was acceptable (< 18%). The relative absorbance value representing the test substance was reduced to 97.9%. This value is well above the threshold for irritation potential (50%). Therefore, under the study conditions, the test substance is considered as not irritating to the skin in a Human Epidermis Skin Model Test (Andres I, 2015a).

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:

February 23, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD Guideline 437 and EU Method B.47, in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

"The measurement of the opacity was performed with a photometer (570 nm) instead of an opacitometer." This deviation was considered as uncritical, because the opacity can be calculated from the absorbance.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

"The measurement of the opacity was performed with a photometer (570 nm) instead of an opacitometer." This deviation was considered as uncritical, because the opacity can be calculated from the absorbance.

GLP compliance:

yes (incl. QA statement)

Species:

other: Bos primigenius Taurus (fresh bovine corneas)

Vehicle:

unchanged (no vehicle)

Controls:

other: in vitro test

Amount / concentration applied:

791.6, 1,063.8 and 1,199.9 mg (applied directly on the cornea using a weight board)

Duration of treatment / exposure:

4 h

Irritation parameter:

other: in vitro irritancy score (IVIS)

Basis:

mean

Time point:

other: 4 h

Score:

5.17

Irritant / corrosive response data:

In the negative control, no signs of eye irritation were observed. The positive control induced serious eye damage, which would be classified as GHS category 1.

The test substance produced effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 5.170.

The experiment is considered as sufficient for the classification of the test substance, because all three replicates of the test substance lead to the same assessment for the test substance.

The absorbance (570 nm) and opacity values which were measured before and after exposition are given in the following table:

Table 1: Absorbance and Opacity Values Negative Control

Parameter	Negative Control
Absorbance before exposition	0.1524	0.1586	0.1575
Absorbance after exposition	0.3357	0.2667	0.2902
Opacity before exposition	1.4204	1.4408	1.4371
Opacity after exposition	2.1662	1.848	1.9507
Opacity Difference	0.7458	0.4072	0.5136

 

Mean opacity difference of the negative control was 0.5555

  

Table 2: Absorbance and Opacity Values Test Substance and Positive Control

Parameter	Test substance			Positive Control
Absorbance before exposition	0.2603	0.1417	0.1195	0.1184	0.1849	0.1239
Absorbance after exposition	0.2702	0.2478	0.2602	1.6291	1.8528	1.7304
Opacity before exposition	1.821	1.3858	1.3167	1.3134	1.5307	1.3301
Opacity after exposition	1.8629	1.7693	1.8205	42.5696	71.2525	53.7527
Opacity Difference	0.042	0.3835	0.5038	41.2562	69.7217	52.4225

For the permeability measurement, three replicates for each treatment group were measured. The optical density values at 490 nm are given in the following table:

Table 3: Optical density at 490 nm

Repl.	Negative Control			Test Substance			Positive Control
Meas.	0.0041	0.0029	0.0028	0.0921	0.0734	0.0609	0.3978	0.2436	0.3568
Corr.	0.0205	0.0145	0.0140	0.4605	0.3670	0.3045	1.9890	1.2180	1.7840
Mean	0.0163			----

Table 2: IVIS

Test group	IVIS	Mean IVIS	Relative standard deviation IVIS
Negative Control 0.9% NaCl	1.053	0.801	28.0%
	0.625
	0.724
Test substance	6.149	5.170	18.2%
	5.088
	4.271
Positive Control 20% imidazole solution	70.3	78.6	10.8%
	87.2
	78.4

Conclusions:

The test substance produced effects on the cornea of the bovine eye with a calculated IVIS (in vitro irritancy score) of 5.170, according to OECD Guideline 437. The experiment is considered as sufficient for the classification of the test substance because all three replicates of the test substance lead to the same assessment (Andres, 2015).

Executive summary:

A study was conducted to assess the corneal damage potential of the test substance in bovine cornea by measuring opacity and permeability according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. The pure test substance was brought onto the cornea of a bovine eye for 4 h at 32 ± 1°C, which had been previously incubated in cMEM, without phenol red, at 32 ± 1°C for 1 h. After removal of the test substance, opacity and permeability values were measured. Under the study conditions, the test substance produced effects on the cornea of the bovine eye with a calculated IVIS (in vitro irritancy score) of 5.170. The negative control (physiological sodium chloride solution) and the positive control (20% imidazole solution) met the validity criteria. The experiment was considered as sufficient for the classification of the test substance as eye irritant because all three replicates of the test substance lead to the same assessment (Andres I, 2015b).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

An in vitro study was conducted to assess the skin irritation potential of the test substance according to OECD Guideline 439 and EU Method B.46, in compliance with GLP. Three tissues of a human skin model EpiDermTM were exposed for 60 minutes. On average, 25 mg of the test substance (wetted with 25 μL DPBS-buffer) were applied to each tissue and spread to match the tissue size (0.63 cm²). DPBS buffer was used as negative control and 5% SDS solution as positive control. After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, with an OD value of 2.3. The positive control showed clear irritating effects with a relative absorbance value reduced to 2.8%. Variation within tissues was acceptable (< 18%). The relative absorbance value representing the test substance was reduced to 97.9%. This value is well above the threshold for irritation potential (50%). Therefore, under the study conditions, the test substance is considered as not irritating to the skin in a Human Epidermis Skin Model Test (Andres I, 2015a).

Eye irritation

A study was conducted to assess the corneal damage potential of the test substance in bovine cornea by measuring opacity and permeability according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. The pure test substance was brought onto the cornea of a bovine eye for 4 h at 32 ± 1°C, which had been previously incubated in cMEM, without phenol red, at 32 ± 1°C for 1 h. After removal of the test substance, opacity and permeability values were measured. Under the study conditions, the test substance produced effects on the cornea of the bovine eye with a calculated IVIS (in vitro irritancy score) of 5.170. The negative control (physiological sodium chloride solution) and the positive control (20% imidazole solution) met the validity criteria. The experiment was considered as sufficient for the classification of the test substance as eye irritant because all three replicates of the test substance lead to the same assessment (Andres I, 2015b).

Justification for selection of skin irritation / corrosion endpoint:
The study was conducted according to internationally accepted guidelines, in compliance with GLP.

Justification for selection of eye irritation endpoint:
The study was conducted according to internationally accepted guidelines, in compliance with GLP.

Effects on eye irritation: irritating

Justification for classification or non-classification

Skin

Based on the available in vivo skin irritation study, the test substance does not require classification for this endpoint according to CLP criteria (EC 1272/2008)

Eye

Based on the available bovine corneal opacity study, the substance is classified as Eye Irrit. 2 (H319: Causes serious eye irritation) according to CLP (EC 1272/2008).