Phosphoric acid, mono- and di-decyl ester, compd. with 2,2',2''-nitrilotris[ethanol]

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Remarks:

In vitro skin corrosion test with EpiDerm (EPI-200))

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 02, 2017 to October 06, 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

Qualifier:

according to guideline

Guideline:

other: EU B.40 BIS "In Vitro Skin Corrosion: Human Skin Model Test

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Remarks:

EpiDerm Skin Model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The 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. Recommended test system in international guidelines (OECD and EC)

Vehicle:

unchanged (no vehicle)

Details on test system:

The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 h before the assay was started the tissues were transferred to 6-well plates containing 0.9 mL DMEM per well. The level of the DMEM was just beneath the tissue. The plates were incubated for approximately 3 h at 37.0 ± 1.0˚C. The medium was replaced with fresh DMEM just before the test substance was applied. The test was performed on a total of 4 tissues per test substance together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test substance and two for a 1 h exposure. 50 µL of the undiluted test substance 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 h time point. After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test substance. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 µl DMEM until 6 tissues (= one application time) were dosed and rinsed.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µL

Duration of treatment / exposure:

3 minutes and 1 h

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Remarks:

cytotoxicity

Run / experiment:

3 minutes treatment

Value:

ca. 71

Vehicle controls validity:

not valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

cytotoxicity

Run / experiment:

1 h treatment

Value:

ca. 103

Vehicle controls validity:

not valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

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

The mean absorption at 570 nm measured after treatment with the test substance and controls were as follows:
1) Negative control 1.988 ± 0.253 (3 minute application) and 1.857 ± 0.050 (1 h application)
2) Test substance 1.407 ± 0.364 (3 minute application) and 1.904 ± 0.197 (1 h application)
3) Positive control 0.333 ± 0.247 (3 minute application) and 0.137 ± 0.028 (1 h application)

- The mean tissue viability obtained after 3 minute and 1 h treatments with the test substance compared to the negative control tissues. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after the 3 minute and 1 h treatments with the test substance compared to the negative control tissues was 71% and 103% respectively. Because the mean relative tissue viability for the test substance was not below 50% after 3 minutes treatment and not below 15% after 1 h treatment the test substance is considered to be not corrosive.

- 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. The mean relative tissue viability following the 1 h exposure to the positive control was 7.4%.

- In the range of 20 - 100% viability the coefficient of variation between tissue replicates was ≤16%, indicating that the test system functioned properly. The coefficient of variation between tissue replicates treated with the test substance was 31% for the 3-minute treatment, which is above acceptance criteria. Since all individual viabilities were >50%, the test outcome was considered valid.

Interpretation of results:

other: CLP criteria not met

Conclusions:

Under the study conditions, the test substance was determined to be non corrosive to skin based on human three dimensional epidermal model (EpiDerm (EPI-200)).

Executive summary:

An in vitro study was conducted to determine the skin corrosion potential of the test substance, Phosphoric acid, mono- and di-decyl ester, compd. with 2,2',2''-nitrilotris[ethanol], according to OECD Guideline 431 and EU Method B.40 bis, in compliance with GLP. The test substance was checked for colour interference in aqueous conditions and possible interference with MTT reduction by adding to MTT medium. Because the solutions did not turn blue/purple nor a blue/purple precipitate was observed, it was concluded that the test substance did not interfere with the MTT. Duplicate tissue samples were exposed to 50 µL test substance for 3 min and 1 h. After the treatment period, a determination of the cytotoxic effect was performed. Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin corrosion was expressed as the remaining cell viability after exposure to the test substance. The positive control potassium hydroxide showed a mean relative tissue viability of 7.4% after the 1-h 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 ≤16%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 3 min and 1 h treatments with the test substance compared to the negative control tissues was 71% and 103%, respectively. Because the mean relative tissue viability for the test substance was above 50 and 15% after 3 min and 1 h treatment respectively, the test substance is considered to be non- corrosive. Under the study conditions, the test substance was determined to be non-corrosive to skin based on human three dimensional epidermal model (EpiDerm 200) (Groot, 2017).

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Remarks:

In vitro skin irritation test with EPISKIN Small Model

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 18, 2017 to November 13, 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)

Version / remarks:

adopted 28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

20 July 2012

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Remarks:

EPISKIN Small ModelTM

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

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. 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:

On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed maintenance medium for 24 h at 37°C. All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 66 - 85%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 37.1°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the humidity and CO2 percentage may occur due to opening and closing of the incubator door. The test was performed on a total of 3 tissues per test substance together with negative and positive controls. 25 µL of the undiluted test substance was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 µL PBS (negative control) and 3 tissues with 25 µL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 h at 37°C.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 µL

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 h at 37°C

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 minutes treatment

Value:

88

Vehicle controls validity:

not valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

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

- The mean absorption at 570 nm measured after treatment with the test substance and controls were as follows:

1) Negative control 1.015 ± 0.067
2) Test substance 0.897± 0.100
3) Positive control 0.071 ± 0.025

- The mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test substance compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test substance compared to the negative control tissues was 88%. Since the mean relative tissue viability for the test substance was above 50% the test substance is considered to be non-irritant.

- The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 7.0%. The absolute mean OD570 of the negative control tissues was with in the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was <10%, indicating that the test system functioned properly.

Interpretation of results:

other: CLP criteria not met

Conclusions:

Under the study conditions, the test substance was determined to be not irritant to skin based on human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SM)).

Executive summary:

An in vitro study was conducted to determine the skin irritation potential of the test substance, Phosphoric acid, mono- and di-decyl ester, compd. with 2,2',2''-nitrilotris[ethanol], according to OECD Guideline 439 and EU Method B.46, in compliance with GLP. The test substance was checked for colour interference in aqueous conditions and possible interference with MTT reduction by adding the test substance to MTT medium. Because the solutions did not turn blue/purple nor a blue/purple precipitate was observed, it was concluded that the test substance did not interfere with the MTT. Triplicate tissue samples were exposed to 25 μL test substance for 15 min. After a 42 h post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation was expressed as the remaining cell viability after exposure to the test substance. The positive control 5% aq. sodium dodecyl sulfate showed a mean relative tissue viability of 7.0% after 15 min exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues were within the laboratory historical control data range. The standard deviation of the percentage viability of three tissues treated identically was < 10%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 ± 0.5 min treatment with the test substance compared to the negative control tissues was 88%. Since the mean relative tissue viability for the test substance was above 50% after 15 ± 0.5 min treatment the test substance is considered to be non-irritant. Under the study conditions, the test substance was determined to be non-irritant to skin based on human three dimensional epidermal model (EPISKIN-SM) (Groot, 2017).

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

The Bovine Corneal Opacity and Permeability Assay (BCOP)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From October 03, 2017 to October 03, 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

Species:

other: Bovine

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 (1-6). 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)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µL

Duration of treatment / exposure:

10 minutes

Observation period (in vivo):

-

Duration of post- treatment incubation (in vitro):

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.

Number of animals or in vitro replicates:

3

Details on study design:

Preparation of corneas:
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum. The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

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. 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.

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 substance 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 substance 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 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.

Irritation parameter:

in vitro irritation score

Run / experiment:

10 minutes exposure

Value:

9.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Inconclusive

Other effects / acceptance of results:

The individual in vitro irritancy scores for the negative controls ranged from 0.2 to 0.7. The individual positive control in vitro irritancy scores ranged from 55 to. The corneas treated with the positive control substance were turbid after the 10 minutes of treatment. The corneas treated with the test substance showed opacity values ranging from 4.0 to 9.8 and permeability values ranging from 0.097 to 0.0315. The corneas were translucent after the 10 minutes of treatment with the test substance. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 8.7 to 11 after 10 minutes of treatment with the test substance.

In Vitro Irritancy Score:

Treatment	Final Opacity^2	Final OD490^2	In vitroIrritancy Score^1
Negative control	0.7	0.000	0.7
	0.3	0.003	0.4
	0.2	0.002	0.2
Positive control	21	2.478	59
	23	2.098	55
	22	2.322	56
The test item	9.8	0.097	11
	7.0	0.139	9.1
	4.0	0.315	8.7

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

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

Summary of Opacity, Permeability and In Vitro Scores:

Treatment	Mean Opacity^1	Mean Permeability^1	MeanIn vitroIrritation Score^1, 2
Negative control	0.4	0.002	0.4
Positive control (Ethanol)	22	2.299	57
The test item	6.9	0.184	9.7

¹  Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.

²  In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD₄₉₀value).

Interpretation of results:

other: Inconclusive

Conclusions:

Under the study conditions, eye damage potential of the test substance was determined to be inconclusive, based on bovine corneal opacity and permeability test (mean IVIS is 9.7).

Executive summary:

An in vitro study was conducted to determine the eye damage potential of the test substance, Phosphoric acid, mono- and di-decyl ester, compd. with 2,2',2''-nitrilotris[ethanol], according to OECD Guideline 437, in compliance with GLP. The test substance was tested through topical application for 10 min. The test substance was applied neat (750 µL) directly on top of the freshly isolated bovine cornea sample. The negative control responses for opacity and permeability was 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 57 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 substance induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 9.7 after 10 min treatment. Under the study conditions, eye damage potential of the test substance was determined to be inconclusive, based on bovine corneal opacity and permeability test (mean IVIS is 9.7) (Groot, 2017).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

EpiOcular™ Cornea Epithelial Model

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From October 23, 2017 to October 27, 2017

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

Species:

human

Details on test animals or tissues and environmental conditions:

Test system details:
The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes. It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test substance to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.

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 eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications (e.g. OECD).

Test system set up:
On the day of receipt the tissues were equilibrated to room temperature. Subsequently, tissues were transferred to 6-well plates and incubated for 20 ± 4 h at 37°C in 1.0 mL fresh pre-warmed assay medium, which was refreshed after approximately 1 h. All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 68 - 85%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.3 - 37.1°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

Application of the test substance:
The test was performed on a total of 2 tissues per test substance together with a negative control and positive control. Before the assay was started the entire tissues were pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS. The tissues were incubated at standard culture conditions for 30 ± 2 minutes. Two tissues were treated with 50 µl Milli-Q water (negative control) and 2 tissues with 50 µL Methyl Acetate (positive control) respectively.
50 µL of the undiluted test substance was added into the 6-well plates on top of the tissues.
After the exposure period with the test substance (30 ± 2 minutes at 37.0 ± 1.0°C), the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test substance. After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 ml of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 12 ± 2 minute immersion incubation at room temperature (Post-Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 ml of warm Assay Medium and were incubated for 120 ± 15 minutes at 37°C.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 µL

Duration of treatment / exposure:

30 ± 2 minutes

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

Duplicate

Irritation parameter:

other: % Viability

Run / experiment:

30 minutes treatment

Value:

4.2

Vehicle controls validity:

not valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Inconclusive

Other effects / acceptance of results:

Interference of the test substance with the MTT endpoint:

The test substance was checked for possible direct MTT reduction by adding the test substance to MTT medium. It observed that test substance did not interact with the MTT endpoint.

The test substance was checked for color interference in aqueous conditions. Addition of the test substance to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of 0.3551 and 0.0001, respectively. Therefore it was concluded that the test substance induced color interference.

The test substance showed color interference in aqueous conditions, in addition to the normal procedure, two tissue were treated with test substance. Instead of MTT solution these tissues were incubated with assay medium. The non-specific color of the test substance was 0.23% of the negative control tissues. The OD of the treated tissues without MTT assay was subtracted from the ODs of the test substance treated viable tissues with MTT assay.

The mean absorption at 570 nm measured after treatment with the test substance and controls were as follows:
1) Negative control 1.768 ± 0.002
2) Test substance 0.075 ± 0.030
3) Positive control 0.664 ± 0.133

The mean tissue viability obtained after 30 ± 2 minutes treatment with the test substance compared to the negative control tissues. Eye hazard potential is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 30 ± 2 minutes treatment with the test substance compared to the negative control tissues was 4.2%. Since the mean relative tissue viability for the test substance was below 60% it is considered to be inconclusive for classification for eye irritation.

The positive control had a mean cell viability after 30 ± 2 minutes exposure of 38%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of two tissues treated identically was less than 8%, indicating that the test system functioned properly.

Interpretation of results:

other: Inconclusive

Conclusions:

Under the study conditions, eye damage potential of the test substance was determined to be inconclusive, based on EpiOcular™ Cornea Epithelial Model (mean viability value is 4.2%).

Executive summary:

An in vitro study was conducted to determine the eye damage potential of the test substance, Phosphoric acid, mono- and di-decyl ester, compd. with 2,2',2''-nitrilotris[ethanol], according to OECD Guideline 492, in compliance with GLP. The test substance was tested through topical application for 30 min. The test substance was applied neat (50 µL) directly on top of the Reconstructed Human EpiOcular™ tissue. After exposure the cornea epithelial construct was thoroughly rinsed to remove the test substance and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 2 h at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect. The positive control methyl acetate showed a mean cell viability of 38% after 30 ± 2 min 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 of the percentage viability of two tissues treated identically was less than 8%, indicating that the test system functioned properly. The test substance showed color interference in aqueous conditions. In addition to the normal procedure, two tissue were treated with test substance. Instead of MTT solution these tissues were incubated with assay medium. The non-specific color of the test substance was 0.23% of the negative control tissues. The OD of the treated tissues without MTT assay was subtracted from the ODs of the treated viable tissues with MTT assay. Eye damage potential is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 30 ± 2 min treatment with the test substance compared to the negative control tissues was 4.2%. Under the study conditions, eye damage potential of the test substance was determined to be inconclusive, based on EpiOcular™ Cornea Epithelial Model (mean viability value is 4.2%) (Groot, 2017).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

Study 1:

An in vitro study was conducted to determine the skin corrosion potential of the test substance, Phosphoric acid, mono- and di-decyl ester, compd. with 2,2',2''-nitrilotris[ethanol], according to OECD Guideline 431 and EU Method B.40 bis, in compliance with GLP. The test substance was checked for colour interference in aqueous conditions and possible interference with MTT reduction by adding to MTT medium. Because the solutions did not turn blue/purple nor a blue/purple precipitate was observed, it was concluded that the test substance did not interfere with the MTT. Duplicate tissue samples were exposed to 50 µL test substance for 3 min and 1 h. After the treatment period, a determination of the cytotoxic effect was performed. Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin corrosion was expressed as the remaining cell viability after exposure to the test substance. The positive control potassium hydroxide showed a mean relative tissue viability of 7.4% after the 1-h 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 ≤16%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 3 min and 1 h treatments with the test substance compared to the negative control tissues was 71% and 103%, respectively. Because the mean relative tissue viability for the test substance was above 50 and 15% after 3 min and 1 h treatment respectively, the test substance is considered to be non- corrosive. Under the study conditions, the test substance was determined to be non-corrosive to skin based on human three dimensional epidermal model (EpiDerm 200) (Groot, 2017).

 

Study 2:

An in vitro study was conducted to determine the skin irritation potential of the test substance, Phosphoric acid, mono- and di-decyl ester, compd. with 2,2',2''-nitrilotris[ethanol], according to OECD Guideline 439 and EU Method B.46, in compliance with GLP. The test substance was checked for colour interference in aqueous conditions and possible interference with MTT reduction by adding the test substance to MTT medium. Because the solutions did not turn blue/purple nor a blue/purple precipitate was observed, it was concluded that the test substance did not interfere with the MTT. Triplicate tissue samples were exposed to 25 μL test substance for 15 min. After a 42 h post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation was expressed as the remaining cell viability after exposure to the test substance. The positive control 5% aq. sodium dodecyl sulfate showed a mean relative tissue viability of 7.0% after 15 min exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues were within the laboratory historical control data range. The standard deviation of the percentage viability of three tissues treated identically was < 10%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 ± 0.5 min treatment with the test substance compared to the negative control tissues was 88%. Since the mean relative tissue viability for the test substance was above 50% after 15 ± 0.5 min treatment the test substance is considered to be non-irritant. Under the study conditions, the test substance was determined to be non-irritant to skin based on human three dimensional epidermal model (EPISKIN-SM) (Groot, 2017).

 

Eye irritation:

Study 1:

An in vitro study was conducted to determine the eye damage potential of the test substance, Phosphoric acid, mono- and di-decyl ester, compd. with 2,2',2''-nitrilotris[ethanol], according to OECD Guideline 437, in compliance with GLP. The test substance was tested through topical application for 10 min. The test substance was applied neat (750 µL) directly on top of the freshly isolated bovine cornea sample. The negative control responses for opacity and permeability was 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 57 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 substance induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 9.7 after 10 min treatment. Under the study conditions, eye damage potential of the test substance was determined to be inconclusive, based on bovine corneal opacity and permeability test (mean IVIS is 9.7) (Groot, 2017).

 

Study 2:

An in vitro study was conducted to determine the eye damage potential of the test substance, Phosphoric acid, mono- and di-decyl ester, compd. with 2,2',2''-nitrilotris[ethanol], according to OECD Guideline 492, in compliance with GLP. The test substance was tested through topical application for 30 min. The test substance was applied neat (50 µL) directly on top of the Reconstructed Human EpiOcular™ tissue. After exposure the cornea epithelial construct was thoroughly rinsed to remove the test substance and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 2 h at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect. The positive control methyl acetate showed a mean cell viability of 38% after 30 ± 2 min 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 of the percentage viability of two tissues treated identically was less than 8%, indicating that the test system functioned properly. The test substance showed color interference in aqueous conditions. In addition to the normal procedure, two tissue were treated with test substance. Instead of MTT solution these tissues were incubated with assay medium. The non-specific color of the test substance was 0.23% of the negative control tissues. The OD of the treated tissues without MTT assay was subtracted from the ODs of the treated viable tissues with MTT assay. Eye damage potential is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 30 ± 2 min treatment with the test substance compared to the negative control tissues was 4.2%. Under the study conditions, eye damage potential of the test substance was determined to be inconclusive, based on EpiOcular™ Cornea Epithelial Model (mean viability value is 4.2%) (Groot, 2017).

Justification for classification or non-classification

Skin irritation:

Based on the results of the in vitro skin irritation studies, Phosphoric acid, mono- and di-decyl ester, compd. with 2,2',2''-nitrilotris[ethanol], does not warrant classification for skin irritation according to EU CLP (Regulation 1272/2008/EC) criteria.

 

Eye irritation:

Results from both the in vitro studies on the test substance produced inconclusive results on their own when interpreted based on the respective OECD guideline criteria. However, considering the evidence together and as a conservative approach, Phosphoric acid, mono- and di-decyl ester, compd. with 2,2',2''-nitrilotris[ethanol], is considered to be irritant to the eyes and warrants a classification as ‘Eye irritant 2 (H319 - causes serious eye irritation) according to EU CLP (Regulation 1272/2008/EC) criteria.