Tall oil, maleated

Endpoint summary

Description of key information

EYE IRRITATION:

EnvaMul 600 induced an IVIS ≤3; therefore, no classification is required for eye irritation or serious eye damage.

SKIN CORROSION:

EnvaMul 600 is considered non corrosive in the in vitro skin corrosion test as:

a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50% (79%)

b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15% (90%).

SKIN IRRITATION:

The relative mean tissue viability obtained after 15 ±0.5 minutes treatment with the test item compared to the negative control tissues was 27%. Since the mean relative tissue viability for EnvaMul 600 was below or equal to 50% after 15 ±0.5 minutes treatment it is considered to be irritant. 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2017-August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Viscous brown liquid

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Cells purchased or derived from tissues obtained by accredited institutions from donor

Source strain:

other: 00267

Justification for test system used:

Recommended test system in international guidelines (OECD and EC)

Vehicle:

unchanged (no vehicle)

Remarks:

The liquid test item was applied undiluted (50 μl) directly on top of the tissue.

Details on test system:

EpiDerm Skin Model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, 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 EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µl undiluted test item was added into the 6-well plates on top of the skin tissues.
For the negative and positive controls, 2 tissues were treated with 50 μl Milli-Q water (negative control) and 2 tissues were treated with 50 μl 8N KOH (positive control) for both the 3-minute and 1-hour time point.

Duration of treatment / exposure:

3 min and 1 hour treatment

Number of replicates:

2 replicates for test item per each exposure time (3 minutes/1 hour)
2 replicates for the positive control per each exposure time (3 minutes/1 hour)
2 replicates for the negative control per each exposure time (3 minutes/1 hour)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 Minute application (first test)

Value:

60

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Since the individual values for the 3-minute exposure were above and below the 50% (71 and 49% respectively) and the Coefficient of Variation was above 30%, the test was inconclusive and a repeat experiment for the 3-minute exposure was performed.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute application (second test)

Value:

79

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour application

Value:

90

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

EnvaMul 600 was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that EnvaMul 600 did not interfere with the MTT endpoint.

Mean Tissue Viability in the in vitro Skin Corrosion Test with EnvaMul 600

First test

3-minute application viability (percentage of control)

Negative control 100

EnvaMul 600 60

Positive control 7.4

Second test

3-minute application viability (percentage of control)

Negative control 100

EnvaMul 600 79

Positive control 9.2

1-hour application viability (percentage of control)

Negative control 100

EnvaMul 600 90

Positive control 5.3

Coefficient of Variation between Tissue Replicates

First test- 3 minute

Negative control 5.1

EnvaMul 600 31

Positive control 26

Second test- 3 minute

Negative control 10

EnvaMul 600 30

Positive control 7.9

1 hour

Negative control 6.8

EnvaMul 600 1.9

Positive control 9.2

CV (%) = 100 - [(lowest OD570/highest OD570) x 100%]

Interpretation of results:

GHS criteria not met

Conclusions:

EnvaMul 600 is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate EnvaMul 600 for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)).

The possible corrosive potential of EnvaMul 600 was tested through topical application for 3 minutes and 1 hour. The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch HD0379UD11 of EnvaMul 600 was a viscous brown liquid. EnvaMul 600 was applied undiluted (50 μl) directly on top of the skin tissue. The positive control had a mean relative tissue viability of 5.3% after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was <7% for the negative control and 31% for the test item. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with EnvaMul 600 compared to the negative control tissues was 60% and 90%, respectively. However, since the individual values for the 3-minute exposure were above and below the 50% (71 and 49% respectively) and the Coefficient of Variation was above 30%, this part of the test was inconclusive and a repeat experiment for the 3-minute exposure was performed.

In the second test, the positive control had a mean relative tissue viability of 9.2% after the 3-minute exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 30%. The relative mean tissue viability obtained after 3-minute treatment with EnvaMul 600 compared to the negative control tissues was 79%. Because the mean relative tissue viability for EnvaMul 600 was not below 50% after the 3-minute treatment in the second experiment and not below 15% after the 1-hour treatment in the first experiment, EnvaMul 600 is considered to be not corrosive. In conclusion, EnvaMul 600 is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

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)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 17-EKIN-038).
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for
13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Source: SkinEthic Laboratories, Lyon, France.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The liquid test item was applied undiluted (25 µl) directly on top of the tissue.

Duration of treatment / exposure:

15 ±0.5 minutes at room temperature

Duration of post-treatment incubation (if applicable):

42 hours at 37 °C

Number of replicates:

3 tissues per test item together with negative and positive controls

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test item (mean tissue viability as % of control)

Value:

27

Negative controls validity:

valid

Remarks:

mean tissue viability (% of control) =100 (SD 7.2)

Positive controls validity:

valid

Remarks:

mean tissue viability (% of control) = 5.4 (SD 1.4)

Remarks on result:

positive indication of irritation

Remarks:

SD 4.4

Irritation / corrosion parameter:

other: mean absorption (optical density)

Run / experiment:

Test item

Value:

0.329

Negative controls validity:

valid

Remarks:

Mean OD 570 = 1.201 (SD 0.087)

Positive controls validity:

valid

Remarks:

OD 570 =0.064 (SD 0.016)

Other effects / acceptance of results:

The test item was checked for possible direct MTT reduction and color interference in the Skin corrosion test using EpiDerm as a skin model (Test Facility Study No. 518198). Because no color changes were observed it was concluded that the test item did not interact with the MTT endpoint.
The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

EnvaMul 600 is irritant in the in vitro skin irritation test under the experimental conditions described in this report and should be classified category 2 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Executive summary:

The objective of this study was to evaluate EnvaMul 600 for its ability to induce skin irritation on a human three-dimensional epidermal model (EPISKIN Small model (EPISKIN-SM^TM)). The possible skin irritation potential of EnvaMul 600 was tested through topical application for 15 minutes.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch HD0379UD11 of the test item was a viscous brown liquid. The test item was applied undiluted (25 µL), directly on top of the skin tissue for 15 ±0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ±0.5 minutes treatment with the test item compared to the negative control tissues was 27%. Since the mean relative tissue viability for EnvaMul 600 was below or equal to 50% after 15 ±0.5 minutes treatment it is considered to be irritant. 

The positive control had a mean cell viability of 5.4% after 15 ±0.5 minutes exposure. The absolute mean OD₅₇₀ (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was <8%, indicating that the test system functioned properly.

In conclusion, EnvaMul 600 is irritant in the in vitro skin irritation test under the experimental conditions described in this report and should be classified as category 2 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

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:

Experimental start date: 23 May 2017, experimental completion date: 23 May 2017

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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Viscous brown liquid

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test System: Bovine eyes were used as soon as possible after slaughter.

Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. As a consequence, a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.

Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.

Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Vehicle:

unchanged (no vehicle)

Remarks:

The test item was heated to 80 ºC to liquefy and was tested neat

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µL of either the negative control, positive control and test item

Duration of treatment / exposure:

10 ±1 min at 32 ±1 °C

Duration of post- treatment incubation (in vitro):

120 ±1 min 32 ±1 °C

Number of animals or in vitro replicates:

3

Details on study design:

Cornea Selection and Opacity Reading
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.
Test Item Preparation
No correction was made for the purity/composition of the test item.
The test item was heated to 80 ºC to liquefy and was tested neat.
Treatment of Corneas and Opacity Measurements
The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control (Ethanol) or test item was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 10 ±1 minutes at 32 ±1 °C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently, the corneas were incubated for 120 ±10 minutes at 32 ±1 °C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

Opacity Measurement
The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
The opacity value (measured with the device OP-KIT) was calculated according to:
Opacity=(I0/I -0.9894)/0.0251
With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea.
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

Application of Sodium Fluorescein
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated.
The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 4 mg Na-fluorescein (Sigma-Aldrich Chemie GmbH, Germany)/ml cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ±5 minutes at 32 ±1°C.

Permeability Determinations
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

Irritation parameter:

cornea opacity score

Run / experiment:

Test Item mean value

Value:

2.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: permeability

Run / experiment:

Test item mean value

Value:

0.015

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

Test item mean value

Value:

2.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

The individual in vitro irritancy scores for the negative controls ranged from -0.4 to 1.2. The individual positive control in vitro irritancy scores ranged from 28 to 48 . The corneas treated with the positive control item were turbid after the 10 minutes of treatment.

The corneas treated with the test item showed opacity values ranging from 1.9 to 2.7 and permeability values ranging from -0.005 to 0.047. The corneas were slightly translucent after the 10 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium. Hence, thein vitro irritancy scores ranged from 1.8 to 3.4 after 10 minutes of treatment with the test item.

 
Opacity Score

Treatment	Opacity before treatment	Opacity after treatment	Final Opacity^1		Negative control corrected Final Opacity^2	Mean Final Opacity
Negative control	1.7	2.9	1.2			0.3
	1.5	1.1	-0.4
	4.4	4.4	0.0
Positive control	-0.5	12.8	13.3	13.0		13
	2.6	17.1	14.4	14.1
	0.7	12.8	12.1	11.8
The test item	1.4	3.7	2.3	2.0		2.2
	0.7	3.7	3.0	2.7
	2.5	4.7	2.2	1.9

1    Final Opacity = Opacity after treatment – Opacity before treatment.

²    Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control. 

³    Calculations are made without rounding off.

 

Table3          
Permeability ScoreIndividual Values (Uncorrected)

Treatment	Dilution factor	OD490 1	OD490 2		OD490 3		Average OD		Final OD		Mean final negative control
Negative control	1	-0.004		-0.004		-0.002		-0.003		-0.003		0.007
	1	0.001		0.002		0.001		0.002		0.002
	1	0.024		0.024		0.022		0.023		0.023
Positive control	1	1.193		1.197		1.182		1.191		1.191
	6	0.382		0.379		0.374		0.378		2.270
	1	1.104		1.084		1.092		1.093		1.093
The test item	1	0.009		0.008		0.009		0.009		0.009
	1	0.054		0.055		0.052		0.054		0.054
	1	0.002		0.003		0.003		0.003		0.003

¹    Calculations are made without rounding off.

  Table4          
Permeability Score Individual Values (Corrected)

Treatment	Dilution factor	Negative control corrected OD4901^1	Negative control corrected OD4902^1	Negative control corrected OD4903^1	Negative control corrected OD490 Average	Negative control corrected final OD490	Average OD
Positive control	1	1.186	1.190	1.175	1.184	1.184	1.499
	6	0.375	0.372	0.367	0.371	2.227
	1	1.097	1.077	1.085	1.086	1.086
The test item	1	0.002	0.001	0.002	0.002	0.002	0.015
	1	0.047	0.048	0.045	0.047	0.047
	1	-0.005	-0.004	-0.004	-0.005	-0.005

¹    OD₄₉₀values corrected for the mean final negative control permeability (0.007).

²    Calculations are made without rounding off.

 

Table5          
In Vitro Irritancy Score

Treatment	Final Opacity^2	Final OD490^2	In vitroIrritancy Score^1
Negative control	1.2	-0.003	1.2
	-0.4	0.002	-0.4
	0.0	0.023	0.3
Positive control	13	1.184	31
	14	2.227	48
	12	1.086	28
The test item	2.0	0.002	2.0
	2.7	0.047	3.4
	1.9	-0.005	1.8

¹  In vitro irritancy score (IVIS) = opacity value + (15 x OD₄₉₀value).

²  Positive control and test item are corrected for the negative control.

 

Interpretation of results:

GHS criteria not met

Conclusions:

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 35 and within two standard deviations of the current historical positive control mean (Appendix 5, Table 6). It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 2.4 after 10 minutes of treatment.
In conclusion, since EnvaMul 600 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
 

Executive summary:

The objective of this study was to evaluate the eye hazard potential of EnvaMul 600 as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).

This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of the test item was tested through topical application for 10 minutes. 

The study procedures described in this report were based on the most recent OECD guideline.

Batch HD0379UD11 of the test item was a viscous brown liquid. The test item was applied as it is (750 µL) directly on top of the corneas.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical rangeindicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 35 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. 

The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 2.4 after 10 minutes of treatment. 

In conclusion, since EnvaMul 600 induced an IVIS ≤3, no classification is required for eye irritation or serious eye damage. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on results from GLP compliant in vitro studies, the substance is to be classified for skin irritation, category 2 but not for eye damage/irritation according to CLP (Regulation EC No 1272/2008).