Phosphoric acid, mono- and di-C6-12-(even numbered)-alkyl esters, potassium salts

Endpoint summary

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:

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

Specific details on test material used for the study:

Chemical name (IUPAC), synonym or trade name: Phosphoric acid, mono- and di-C6-12-(even numbered)-alkyl esters, potassium salts
Appearance: White solid
Batch: RE 14-6
Purity/Composition: UVCB
Test substance storage: At room temperature
Stable under storage conditions until: 27 July 2018 (expiry date)

Test system:

human skin model

Remarks:

EpiDerm Skin Model

Source species:

human

Cell type:

other: human-derived epidermal 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 hour 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 hours at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM just before the test item was applied. The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure. 50 µL of the 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. After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item. 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

Duration of treatment / exposure:

3 minutes and 1 h

Number of replicates:

4 tissues per test susbtance two tissues were used for a 3-minute exposure and two for a 1-h exposure.

Irritation / corrosion parameter:

other: remaining cell viability after exposure to the test item

Run / experiment:

3 minutes

Value:

ca. 88

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

other: remaining cell viability after exposure to the test substance

Run / experiment:

1 h

Value:

ca. 106

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:

Test substance 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 the test item did not interfere with the MTT endpoint.

The mean absorption at 570 nm measured after treatment with the test substance and controls are presented in Table 1 in below sections. The individual OD570 measurements are presented in table 2.

Table 3 shows the mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 88% and 106% respectively. Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the test item 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-hour 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.

Table1: Mean Absorption in thein vitro skin corrosion test with test substance

	3-minute application					1-hour application
	A (OD570)	B (OD570)	Mean (OD570)		SD	A (OD570)	B (OD570)	Mean (OD570)		SD
Negative control	1.810	2.167	1.988	±	0.253	1.822	1.893	1.857	±	0.050
Test item	1.791	1.696	1.744	±	0.067	2.082	1.842	1.962	±	0.170
Positive control	0.508	0.158	0.333	±	0.247	0.156	0.117	0.137	±	0.028

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.0431). Isopropanol was used to measure the background absorption.

Table 2: Individual OD Measurements at 570 nm

	3-minute application (OD570)        A               B		1-hour application (OD570)        A               B
Negative control OD570measurement 1 OD570measurement 2 OD570measurement 3
	1.8603	2.2101	1.8066	1.9555
	1.8617	2.2242	1.9189	1.9235
	1.8367	2.1960	1.8688	1.9290
Test item OD570measurement 1 OD570measurement 2 OD570measurement 3
	1.8565	1.7476	2.1751	1.8878
	1.8369	1.7341	2.1298	1.8969
	1.8095	1.7360	2.0712	1.8701
Positive control OD570measurement 1 OD570measurement 2 OD570measurement 3
	0.5508	0.2007	0.2011	0.1605
	0.5511	0.2017	0.1988	0.1604
	0.5525	0.2020	0.1984	0.1601

OD = Optical density

Duplicate exposures are indicated by A and B.

Table 3: Mean Tissue viability in the in vitro skin corrosion test with test substance

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
Test item	88	106
Positive control	17	7.4

Table 4: Coefficient of variation between tissue replicates

	3 minute	1 hour
Negative control	16	3.8
Test item	5.3	12
Positive control	69	25

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

Interpretation of results:

other: CLP criteria not met

Conclusions:

Under the study conditions, the test substance was considered to be non- corrosive to human-derived epidermal keratinocytes (EpiDerm Skin Model).

Executive summary:

A study was conducted to determine the in vitro skin corrosion potential of the test substance, according to OECD Guideline 431and EU Method B.40 BIS: "In VitroSkin Corrosion: Human Skin Model Test (EpiDerm Skin Model)in compliance with GLP. The test substance was checked for colour 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 the test item did not interfere with the MTT endpoint. Duplicates were exposed to test substance for 3 minutes and 1 hour. Post 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 had a mean relative tissue viability of 7.4% 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 ≤16%, indicating that the test system functioned properly. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test substance compared to the negative control tissues was 88% and 106%, respectively.Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the test item is considered to be not corrosive. Under the study conditions, the test substance was considered to be non- corrosive to human-derived epidermal keratinocytes (EpiDerm Skin Model) (Groot, 2017).

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 07, 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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Identification: 0312 PHOSPHORSÄUREPARTIALESTER K-SALZ C8-10
- Appearance: White solid
- Batch: RE 14-6
- Purity/Composition: UVCB
- Test substance storage: At room temperature
- Stable under storage conditions until: 27 July 2018 (expiry date)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Adult human-derived epidermal keratinocytes

Details on animal used as source of test system:

EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 17-EKIN-045): 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 d, 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.

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)

Remarks:

The test substance has a waxy consistence and was applied directly on top of the skin tissue and spread to match the size of the tissue.

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 23.5 hours at 37°C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.

Environmental condition:
All incubations, with the exception of the test susbtance 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. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

55.46 to 91.91 mg

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours at 37°C

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 minutes exposure

Value:

12

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Mean absorption in the in vitro skin irritation test with the test substance:

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)		SD
Negative control	1.090	0.962	0.991	1.015	±	0.067
The test item	0.149	0.095	0.110	0.118	±	0.028
Positive control	0.052	0.100	0.060	0.071	±	0.025

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.042). Isopropanol was used to measure the background absorption.

 
Mean tissue viability in the in vitro skin irritation test with the test substance:

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	6.6
The test item	12	2.8
Positive control	7.0	2.5

 

Interpretation of results:

other: Category 2 (irritant) based on CLP criteria

Conclusions:

Under the study conditions, the test substance was determined to be irritant (category 2) to skin based on human skin model (EPISKIN-SMTM).

Executive summary:

A study was conducted to determine the in vitro skin irritation potential of the test substance 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 sample were exposedat least 10 mgof the test substance (55.46 to 91.91 mg) was applied to the tissue, which was pre-moistened with 5 μL water to ensure good contact with the tissue surface for 15 minutes. Post 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 irritation was expressed as the remaining cell viability after exposure to the test substance. The positive control had a mean relative tissue viability of 7.0% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 439 and the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 7.0%, indicating that the test system functioned properly.The relative mean tissue viability obtained after 15 minutes treatment with the test substance compared to the negative control tissues was 12%. Because the mean relative tissue viability for the test substance was below 50% after 15 minute treatment, so the test substance is considered to be irritant (category 2). Under the study conditions, the test substance was determined to be irritant (category 2) to skin based on human skin model (EPISKIN-SM^TM) (Groot, 2017).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

The Bovine Corneal Opacity and Permeability Assay (BCOP)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 09, 2017 to October 10, 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:

yes

Remarks:

Deviations does not impact the outcome of the study

GLP compliance:

yes

Specific details on test material used for the study:

Appearance: White solid
Batch: RE 14-6
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until: 27 July 2018 (expiry date)

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:

303.7 to 363.6 mg

Duration of treatment / exposure:

4 h

Observation period (in vivo):

-

Duration of post- treatment incubation (in vitro):

90 minutes

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 (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Fetal Bovine Serum (Life Technologies)). 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°C ± 1°C. The corneas were incubated for the minimum of 1 hour at 32°C ± 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 (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 will be made for the purity/composition of the test compound. Since no workable suspension of the test item in physiological saline could be obtained, the test item was used as delivered by the sponsor and added pure on top of the corneas.

Treatment of Corneas and Opacity Measurements:
The medium from the anterior compartment was removed and 750 µL of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. The test item was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (303.7 to 363.6 mg). The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32°C ± 1°C. After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies). Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

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 5 mg Na-fluorescein/ml cMEM solution (Sigma-Aldrich Chemie GmbH, Germany).
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°C ±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:

in vitro irritation score

Run / experiment:

4 h exposure

Value:

126

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Positive indication of corrosive

Other effects / acceptance of results:

The individual in vitro irritancy scores for the negative controls ranged from 0.8 to 4.7. One of the negative control eyes was excluded from the analysis since the permeability was above the historical data base which resulted in an IVIS outside the historical data base. Since the other two eyes completely met the criteria and the test substance results were not influenced by this result, this does not affect the study outcome. The individual positive control in vitro irritancy scores ranged from 112 to 199. The corneas treated with the positive control were turbid after the 240 minutes of treatment. The corneas treated with the test item showed opacity values ranging from 25 to 42 and permeability values ranging from 5.680 to 6.980. The corneas were turbid after the 240 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 110 to 134 after 240 minutes of treatment with the test substance.

Table1: Summary of opacity, permeability and in vitro scores

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score^1, 2
Negative control	2.5	0.021	2.8
Positive control	123	1.758	150
Test item	32	6.253	126

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 OD490 value).

Table 2: Individual Opacity, Permeability and in vitro Scores

Treatment	Opacity before treatment	Opacity after treatment	Final Opacity^1		Negative control corrected Final Opacity^2	Mean Final Opacity
Negative control	4.8	5.5	0.7			2.5
	4.5^4	8.6^4	4.0^4
	3.9	8.1	4.2
Positive control	3.5	183.0	179.5	177		123
	3.3	93.2	89.9	87
	3.4	111.0	107.6	105
Test item	5.0	49.6	44.6	42		32
	4.4	36.6	32.1	30
	6.5	34.2	27.7	25

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.

⁴    Excluded from analysis (see study plan deviation).

Table 3: Permeability Score Individual Values (Corrected)

Treatment	Dilution factor	Negative control corrected OD490 1^1	Negative control corrected OD490 2^1	Negative control corrected OD490 3^1	Negative control corrected OD490 Average	Negative control corrected final OD490	Average OD
Positive control	1	1.423	1.424	1.453	1.433	1.433	1.758
	6	0.281	0.277	0.277	0.278	1.670
	6	0.363	0.365	0.358	0.362	2.172
Test item	6	1.023	1.006	1.020	1.016	6.098	6.253
	6	1.172	1.160	1.158	1.163	6.980
	6	0.955	0.944	0.941	0.947	5.680

¹  OD490 values corrected for the mean final negative control permeability (0.021).

²    Calculations are made without rounding off.

Table 4: In VitroIrritancy Score

Treatment	Final Opacity^2	Final OD490^2	In vitroIrritancy Score^1
Negative control	0.7	0.007	0.8
	4.0^3	0.558^3	12^3
	4.2	0.035	4.7
Positive control	177	1.433	199
	87	1.670	112
	105	2.172	138
Test item	42	6.098	134
	30	6.980	134
	25	5.680	110

¹  In vitro irritancy score (IVIS) = opacity value + (15 x OD490 value).

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

³    Excluded from analysis (see study plan deviation).

Interpretation of results:

other: Category 1 (irreversible effects on the eye) based on CLP criteria

Conclusions:

Under the study conditions, the test substance was considered to be causing serious eye damage (category 1) to Bovine Corneal Opacity and Permeability Test (BCOP Test) (IVIS-126).

Executive summary:

A study was conducted to determine the in vitro eye damage potential of the test substance, according to OECD Guideline 437 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), in compliance with GLP. The eye damage of the test substance was tested through topical application for 4 h. The study procedures described in this report were based on the most recent OECD guideline. The test substance was a white solid. The test substance was applied as it is (303.7 to 363.6 mg) directly on top of the corneas. 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 individual positive control in vitro irritancy scores ranged from 112 to 199. The corneas treated with the positive control were turbid after the 4 h of treatment. The corneas treated with the test substance showed opacity values ranging from 25 to 42 and permeability values ranging from 5.680 to 6.980. The corneas were turbid after the 240 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 110 to 134 after 240 minutes of treatment with the test substance. Under the study conditions, the test substance was considered to be causing serious eye damage (category 1) to Bovine Corneal Opacity and Permeability Test (BCOP Test) (IVIS-126) (Groot, 2017).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin corrosion:

A study was conducted to determine the in vitro skin corrosion potential of the test substance according to OECD Guideline 431and EU Method B.40 BIS: "In VitroSkin Corrosion: Human Skin Model Test (EpiDerm Skin Model)in compliance with GLP. The test substance was checked for colour 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 the test item did not interfere with the MTT endpoint. Duplicates were exposed to test substance for 3 minutes and 1 hour. Post 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 had a mean relative tissue viability of 7.4% 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 ≤16%, indicating that the test system functioned properly. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test substance compared to the negative control tissues was 88% and 106%, respectively.Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the test item is considered to be not corrosive.Under the study conditions, the test substance was considered to be non- corrosive to human-derived epidermal keratinocytes (EpiDerm Skin Model) (Bachelor, 2017).

Skin irritation:

A study was conducted to determine the in vitro skin irritation potential of the test substance 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 sample were exposedat least 10 mgof the test substance (55.46 to 91.91 mg) was applied to the tissue, which was pre-moistened with 5 μL water to ensure good contact with the tissue surface for 15 minutes. Post 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 irritation was expressed as the remaining cell viability after exposure to the test substance. The positive control had a mean relative tissue viability of 7.0% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 439 and the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 7.0%, indicating that the test system functioned properly.The relative mean tissue viability obtained after 15 minutes treatment with the test substance compared to the negative control tissues was 12%. Because the mean relative tissue viability for the test substance was below 50% after 15 minute treatment, so the test substance is considered to be irritant (category 2). Under the study conditions, the test substance was determined to be irritant (category 2) to skin based on human skin model (EPISKIN-SM^TM) (Groot, 2017).

Eye irritation:

A study was conducted to determine the in vitro eye damage potential of the test substance according to OECD Guideline 437 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), in compliance with GLP. The eye damage of the test substance was tested through topical application for 4 h. The study procedures described in this report were based on the most recent OECD guideline. The test substance was a white solid. The test substance was applied as it is (303.7 to 363.6 mg) directly on top of the corneas. 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 individual positive control in vitro irritancy scores ranged from 112 to 199. The corneas treated with the positive control were turbid after the 4 h of treatment. The corneas treated with the test substance showed opacity values ranging from 25 to 42 and permeability values ranging from 5.680 to 6.980. The corneas were turbid after the 240 minutes of treatment with the test substance. No pH effect of the test substance was observed on the rinsing medium. Hence, thein vitroirritancy scores ranged from 110 to 134 after 240 minutes of treatment with the test substance. Under the study conditions, the test substance was considered to be causing serious eye damage (category 1) to Bovine Corneal Opacity and Permeability Test (BCOP Test) (IVIS-126) (Bachelor, 2017).

Justification for classification or non-classification

Based on the results of in vitro skin corrosion, in vitro skin irritation and in vitro eye irritation studies, the substance warrants skin irritant 2 (H315: Causes skin irritation) and eye damage 1 1 (H318: Causes serious eye damage) classification according to CLP (EC 1272/2008) criteria.