Tall oil, compd. with ethanolamine

Endpoint summary

Description of key information

SKIN IRRITATION
In an in vitro study, the read across material Fatty acids, tall-oil, compds. with ethanolamine was determined to be a non irritant.
EYE IRRITATION
In an in vitro study, the test material was determined to be a non irritant.

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:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

16 August 2010 to 16 September 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

The study was conducted in accordance with the draft guideline OECD Guideline for the Testing of Chemicals, Draft Proposal for a New Guideline, In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method, Version 4, 11 December 2009.

GLP compliance:

yes (incl. QA statement)

Species:

other: in vitro Reconstructed Human Epidermis (RHE) Model

Details on test animals or test system and environmental conditions:

The EpiDerm EPI-200 skin model consists of normal human epidermal keratinocytes (NHEK) from one single donor, derived from neonatal-foreskin tissue. The keratinocytes are plated on chemically modified, collagen-coated, 8 mm ID cell culture inserts (surface area 0.5 cm²). The skin models are commercially available.
Upon arrival at the testing laboratory, the EPI-200 skin models were pre-incubated in 0.9 mL assay medium provided with the kit for 60 minutes (at ca. 37 °C and ca. 5 % CO₂). At the end of the first pre-incubation period, the tissues were additionally pre-incubated overnight for 21 to 24 hours (at ca. 37 °C and ca. 5 % CO₂).

EPI-200 skin model supplier: MatTek Corporation, USA

Type of coverage:

other: not applicable

Preparation of test site:

other: not applicable

Vehicle:

unchanged (no vehicle)

Controls:

other: in vitro controls were used

Amount / concentration applied:

30 µL

Duration of treatment / exposure:

A treatment period of 60 minutes was followed by a post-exposure incubation period of 42 hours.

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

REFERENCE MATERIALS
- Positive control: 5 % Sodium Lauryl Sulphate (SLS) in demineralised water (= 5 % Sodium Dodecyl Sulphate (SDS))
- Negative control: Phosphate Buffered Saline (PBS)

PRELIMINARY TEST
The tissue staining potential of the test material was investigated prior to the main study by incubating 30 µL in 300 µL of demineralised water for 1 hour (at ca. 37 °C). At the end of the exposure time, the presence and intensity of the staining (if any) was evaluated. The test material did not change the colour of the solution; therefore, it was concluded that the test material did not have the potential to stain the tissue.
Some chemicals are known to non-specifically reduce MTT, resulting in a blue precipitate. Therefore, prior to the start of the main study, 30 µL of the test material was incubated in 1 mL of 1 mg/mL MTT solution for 3 hours (at ca. 37 °C) and the formation of a blue formazan product was assessed. The test material did not show MTT reducing capacity, i.e. the solution did not turn blue/purple.
Furthermore, the test material showed no interaction with a nylon mesh. Therefore, nylon mesh was used to facilitate equal distribution of the test material.

EXPOSURE TO THE TEST MATERIAL
After overnight pre-incubation, the skin membranes were exposed to 30 µL of the test material, positive or negative controls in triplicate for 60 ± 2 minutes. The test material was manually shaken just before application to facilitate homogenisation. Immediately after application, a nylon mesh (provided with the EPI-200 kit) was placed onto the tissue surface to facilitate an equal distribution of the test material.

Exposure was performed at ambient temperature in a flow cabin in four series, including one series of frozen controls. After dosing the last tissue of each series, the plates containing the skin membranes were transferred to a humidified incubator (ca. 37 °C and ca. 5 % CO₂). After 35 ± 1 minutes, the plates were removed from the incubator and placed in the flow cabin until the exposure period of 60 minutes was completed for the first dosed tissue. When the exposure period was completed, the inserts were removed from the well and the skin surface was carefully washed using an excess of phosphate buffered saline (PBS).
Subsequently, the insert was blotted dry and the tissue surface was carefully dried using a sterile cotton swab. The insert was then transferred to clean 6-well plate containing fresh medium (0.9 mL/well). Medium was refreshed after 18 ±2 hours. Following an additional 22.5 hours incubation period at ca. 37 °C and ca. 5 % CO₂ in a humidified incubator, viability was determined using the MTT assay.

MTT ASSAY
An MTT solution of 1 mg/mL was prepared by diluting MTT concentrate 5 times in MTT diluent (both provided with the kit). The bottom of the inserts was blotted dry and the inserts placed in a 24-well plate containing 300 µL of MTT medium/well. The plates were placed in an incubator at ca. 37 °C and ca. 5 % CO₂. After 180 ± 1 minutes, the tissues were rinsed three times with PBS. The formazan product was extracted from the tissue using 2 mL MTT extractant (provided with the kit). Extraction was performed in the dark for 2 days at 2 to 10 °C.

After completion of the extraction period, the skin membrane was pierced with a needle to allow the extract to run into the well from which the membrane was taken. The optical density was measured in triplicate in 200 µL sub-fractions using a spectrophotometer set at 570 nm. Extractant solvent alone was used as blank. The mean optical density was calculated and expressed as the percentage viability compared to the negative control.

INTERPRETATION OF THE RESULTS
The test is considered valid if the optical density of the negative control is ≥1.0 and ≤2.5 and the optical density of the extractant solvent alone is <0.1. Tissues treated with the positive control should reflect their ability to respond to an irritant chemical.

The irritation potential of the test material was determined from the relative mean tissue viability compared to the negative control tissues, using the following prediction model:

Mean tissue viability in vitro In vivo prediction
(% of negative control)

Mean tissue viability ≤50 % Irritant
Mean tissue viability >50 % Non-irritant

Irritant / corrosive response data:

The results are summarised in Table 1.
The mean optical density of the test material was 94 % of the negative control.

The optical densities of the negative and positive control were within the acceptable ranges and correctly indicated non irritancy and irritancy, respectively. This demonstrated validity of the model.

Table 1 Summary of Control and Test Material Results

Material			MTT Conversion (OD₅₇₀)
			Measurement 1	Measurement 2	Measurement 3	Replicate Mean
Negative Control	Replicate 1		1.924	1.921	1.983	1.943
	Replicate 2		1.980	1.978	1.995	1.984
	Replicate 3		1.933	1.975	1.970	1.959
	Mean	1.962
	SD	0.021
	% CV	1.1
	% of control	100
Test Material	Replicate 1		2.060	2.090	2.059	2.070
	Replicate 2		1.611	1.643	1.630	1.628
	Replicate 3		1.828	1.861	1.837	1.842
	Mean	1.847
	SD	0.221
	% CV	12.0
	% of control	94
Positive Control	Replicate 1		0.127	0.136	0.138	0.134
	Replicate 2		0.137	0.137	0.135	0.136
	Replicate 3		0.133	0.132	0.133	0.133
	Mean	0.134
	SD	0.002
	% CV	1.4
	% of control	7

 Data shown in the table were corrected for background absorbance values (extractant solvent alone), which were on average 0.040.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the test material was determined to be a non irritant in accordance with EU criteria.

Executive summary:

The potential of the test material to cause skin irritation was assessed in an in vitro study conducted under GLP conditions in accordance with the draft guideline “OECD Guideline for the Testing of Chemicals, Draft Proposal for a New Guideline, In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method, Version 4, 11 December 2009”.

The study used the EpiDerm reconstituted skin membranes. The undiluted test material was applied topically to the skin membranes for 60 minutes. After a 42 hour incubation period, the effect on the tissue viability was determined, based on the reduction of MTT to a purple formazan precipitate. 5 % Sodium Dodecyl Sulphate (SDS) was used as the positive control and Phosphate Buffered Saline (PBS) as the negative.

The mean optical density of the test material was 94 % of the negative control.

The optical densities of the negative and positive control were within the acceptable ranges and correctly indicated non irritancy and irritancy, respectively. This demonstrated validity of the model.

Under the conditions of this study, the test material was determined to be a non irritant and requires no classification in accordance with EU criteria.

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:

27 July 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: Bovine cornea

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Eyes from adult cattle were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee and placed in Hanks' Balanced Salt Solution (HBSS), supplemented with Penicillin/Streptomycin, and transported to the laboratory on ice packs. The eyes were refrigerated on arrival and used within 24 hours of receipt.
- Preparation of Corneas: The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed (epithelial side uppermost) in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete minimum essential medium (MEM) and plugged. The holders were incubated at 32 ± 1 °C for 60 minutes.

Vehicle:

unchanged (no vehicle)

Controls:

other: concurrent positive and negative controls

Amount / concentration applied:

0.75 mL

Duration of treatment / exposure:

The undiluted test material was applied for 10 minutes, followed by an incubation period of 120 minutes.

Number of animals or in vitro replicates:

3 corneas were exposed to the test material.

Details on study design:

SELECTION OF CORNEAS AND OPACITY READING
After preparation of the corneas, the medium from both chambers of each holder was replaced with fresh complete MEM.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.

TREATMENT OF CORNEAS
The MEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test or control materials were applied to the cornea. 0.9 % w/v sodium chloride solution was used as supplied as the negative control; ethanol was used undiluted as supplied as the positive control. The holders were gently tilted back and forth to ensure a uniform application of the material over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 °C for 10 minutes.
At the end of the exposure period, the test and control materials were removed from the anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM. The anterior chamber was refilled with fresh complete MEM. A post-treatment opacity reading was taken and each cornea was visually observed.
The holders were incubated, anterior chamber facing forward, at 32 ± 1 °C for 120 minutes ± 10 minutes.
After incubation the holders were removed from the incubator and a final opacity reading was taken. Each cornea was visually observed.

APPLICATION OF SODIUM FLUORESCEIN
Following the opacity measurement, the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 °C for 90 minutes.

PERMEABILITY DETERMINATIONS
After incubation, the medium in the posterior chamber of each holder was decanted and retained.
360 µL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured.
If values greater than 1.500 OD492 were obtained, a 1 in 5 dilution of the medium in complete MEM was performed and the measurement repeated. The modified value was multiplied by 5 to reflect the 1 in 5 dilution.

HISTOPATHOLOGY
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labelled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10 % neutral buffered formalin.

EVALUATION OF RESULTS
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.
-Opacity Measurement
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
-Permeability Measurement
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.
-In Vitro Irritancy Score
The following formula was used to determine the In Vitro Irritancy Score:
In Vitro Irritancy Score = mean opacity value + (15 x mean OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test material induced a response through only one of the two endpoints.
-Visual Observation
The condition of the cornea was visually assessed immediately after rinsing and at the final opacity measurement.

DATA INTERPRETATION
A test material that induces an In Vitro Irritancy Score ≥55.1 is defined as an ocular corrosive or severe irritant.

CRITERION FOR AN ACCEPTABLE TEST
For an acceptable test the following positive control criterion must be achieved:
The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean for the testing facility. Ethanol was used for positive control purposes and the In Vitro Irritancy Score should fall within the range of 30.9 to 67.7.

Irritation parameter:

other: In Vitro Irritancy Score

Basis:

mean

Remarks:

of three treated corneas

Score:

21.8

Irritant / corrosive response data:

CORNEAL OPACITY AND PERMEABILITY MEASUREMENT
Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in Table 1.

CORNEAL EPITHELIUM CONDITION
The condition of each cornea post treatment and at the final opacity measurement is given in Table 2.

IN VITRO IRRITANCY SCORE
The results are summarised as follows:
Test material: 21.8
Negative Control: 0.7
Positive Control: 33.9

Table 1 Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity					Permeability (OD)		In Vitro Irritancy Score
		Pre-treatment	Post-treatment	Post-incubation	Post-incubation Minus Pre-treatment	Corrected Value		Corrected Value
Negative Control	1	4	3	4	0		0.022
	2	4	3	5	2		0.024
	3	3	2	2	-1		0.025
					0.3*		0.024‡		0.7
Positive Control	4	2	26	26	24	23.7	0.677	0.653
	5	2	28	29	27	26.7	0.545	0.521
	6	1	23	29	28	27.7	0.434	0.410
						26.0†		0.528†	33.9
Test Material	7	3	10	17	14	13.7	0.508	0.484
	8	3	11	21	18	17.7	0.551	0.527
	9	4	17	16	12	11.7	0.509	0.485
						14.3†		0.499†	21.8

OD = Optical density

*Mean of the post-incubation minus pre-treatment values

†Mean corrected value

‡Mean permeability

 

Table 2 Corneal Epithelium Condition Post-treatment and Post-incubation

Treatment	Cornea Number	Observation
		Post-treatment	Post-incubation
Negative Control	1	Clear	Clear
	2	Clear	Clear
	3	Clear	Clear
Positive Control	4	Cloudy	Cloudy
	5	Cloudy	Cloudy
	6	Cloudy	Cloudy
Test Material	7	Slightly cloudy	Slightly cloudy
	8	Slightly cloudy	Slightly cloudy
	9	Slightly cloudy	Slightly cloudy

Interpretation of results:

other: Not a severe irritant or corrosive

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The test material is not a severe irritant or corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions in this study in accordance with EU criteria.

Executive summary:

The corrosion and irritancy potential of the test material was investigated in vitro using the Bovine Corneal Opacity and Permeability Assay (BCOP) in accordance with the standardised guideline OECD 437 under GLP conditions.

The undiluted test material was applied for 10 minutes, followed by an incubation period of 120 minutes. Negative (0.9 % w/v sodium chloride solution) and positive controls (ethanol) were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The IVIS for the test material was found to be 21.8.

Under the conditions of this study, the test material was considered not to be an ocular corrosive or severe irritant and as such requires no classification in accordance with EU criteria.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation

The potential of the read across material Fatty acids, tall-oil, compds. with ethanolamine to cause skin irritation was assessed in an in vitro study conducted under GLP conditions in accordance with the draft guideline “OECD Guideline for the Testing of Chemicals, Draft Proposal for a New Guideline, In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method, Version 4, 11 December 2009”. It was assigned a reliability score of 2 in accordance with the criteria detailed by Klimisch (1997).

The study used the EpiDerm reconstituted skin membranes. The undiluted test material was applied topically to the skin membranes for 60 minutes. After a 42 hour incubation period, the effect on the tissue viability was determined, based on the reduction of MTT to a purple formazan precipitate. 5 % Sodium Dodecyl Sulphate (SDS) was used as the positive control and Phosphate Buffered Saline (PBS) as the negative.

The mean optical density of the test material was 94 % of the negative control.

The optical densities of the negative and positive control were within the acceptable ranges and correctly indicated non irritancy and irritancy, respectively. This demonstrated validity of the model.

Under the conditions of this study, the test material was determined to be a non irritant and requires no classification in accordance with EU criteria. On this basis, it is considered that Tall Oil, compound with ethanolamine will be non irritating also.

 

Eye Irritation

The corrosion and irritancy potential of the test material was investigated in vitro using the Bovine Corneal Opacity and Permeability Assay (BCOP) in accordance with the standardised guideline OECD 437 under GLP conditions. It was assigned a reliability score of 1 in accordance with the criteria detailed by Klimisch (1997).

The undiluted test material was applied for 10 minutes, followed by an incubation period of 120 minutes. Negative (0.9 % w/v sodium chloride solution) and positive controls (ethanol) were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The IVIS for the test material was found to be 21.8.

Under the conditions of this study, the test material was considered not to be an ocular corrosive or severe irritant and as such requires no classification in accordance with EU criteria.

  

Further information is provided in the form of a study on the read across material Fatty acids, tall-oil, compds. with ethanolamine. The potential of the undiluted test material to cause corrosive or severe irritancy effects to the eye was investigated in vitro in an Isolated Chicken Eye (ICE) study conducted in accordance with the standardised guideline OECD 438 under GLP conditions. It was assigned a reliability score of 2 in accordance with the criteria detailed by Klimisch (1997).

The isolated chicken eyes were exposed to the test material for 10 seconds followed by a 20 mL saline rinse. The eyes were evaluated after 30, 75, 120, 180 and 240 minutes for the adverse eye effects of corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells. Physiological saline (0.9 %) was used as negative control whilst benzalkonium chloride (as a 5 % w/w aqueous solution) served as positive control.

The test material caused very slight corneal swelling, slight corneal opacity and very slight fluorescein retention. The negative control eyes did not show any corneal effect and demonstrated that the general conditions during the test were adequate. The positive control eyes showed severe corneal effects (calculated Irritation Indices ranged from 152 to 155) and demonstrated the suitability and sensitivity of the ICE to detect severe eye irritants.

Under the conditions of this study, the test material did not cause any corrosive or severe irritancy effects and as such requires no classification in accordance with EU criteria. On this basis, it is considered that Tall Oil, compound with ethanolamine will be non irritating also. Given that the potential of the substance is adequately addressed through the use of in vitro studies, it was considered unjustified to conduct in vivo testing.

Justification for selection of skin irritation / corrosion endpoint:
One study available.

Justification for selection of eye irritation endpoint:
The key study was carried out in accordance with the standardised guideline OECD 437 under GLP conditions. It was assigned a reliability score of 1 in accordance with the criteria detailed by Klimisch (1997).

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation 1272/2008, the test material does not require classification for irritation or corrosion to the skin and eye.