Aluminum magnesium oxide

Endpoint summary

Description of key information

Skin irritation (OECD TG 439): not irritating

Skin corrosion (OECD TG 431: not corrosive

Eye irritation (OECD TG 437): serious eye damage category 1

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 August 2018 - 03 September 2018

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:

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

Source species:

human

Cell type:

other: epidermal keratinocytes

Cell source:

other: SkinEthic Laboratories, Lyon, France.

Source strain:

other: not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM(EPISKIN-SMTM, 0.38 cm^2
- Tissue batch number(s): 18 EKIN 035

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing: phosphate buffered saline
- Time after start of exposure: 15 minutes

TEST FOR DIRECT MTT REDUCTION AND COLOUR INTERFERENCE
KW-2200 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. 20153229).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Microplate reader: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 exposure time.
EVALUATION
The corrected OD (ODc) for each sample or control will be calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw).
ODc = ODraw – ODbl
The OD value representing 100% cell viability is the average OD of the negative controls (ODlt_u+MTT).
The %Viability for each sample and positive control is calculated as follows:
%Viability = (ODc/mean ODlt_u+MTT) * 100

PREDICTION MODEL / DECISION CRITERIA: see Table 1

ACCEPTABILITY CRITERIA:
The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be <=18.
b) The mean relative tissue viability of the positive control should be <=40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be <=18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be <=18.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amounts applied: 12.1 to 13.0 mg, the skin was moistened with 5 μL Milli-Q water.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 minutes exposure

Value:

101

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

26

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control tissues was within the laboratory historical control data range.
- Acceptance criteria met for positive control: The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 26%.
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically was < 8%, indicating that the test system functioned
properly.

Interpretation of results:

other: Not skin irritating.

Remarks:

According to Regulation (EC) No. 1272/2008 and its amendments.

Conclusions:

The in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles. Since the mean relative tissue viability for KW-2200 was above 50%, KW-2200 is considered to be non-irritant.

Executive summary:

The substance was tested in triplicate in an in vitro skin irritation test according to OECD TG 439 test guideline and GLP principles. Tissues were exposed to KW-2200, a negative control (PBS) and a positive control (5% SDS) for 15 minutes. The substance was tested for direct MTT reduction and colour interference and both results were negative. Acceptability criteria for the negative control, positive control and variability between measurements were met.

The cell viability of the tissues exposed to KW-2200 was 101% for 15 minutes exposure. Since the mean relative tissue viability for KW-2200 was above 50%, KW-2200 is considered to be non-irritant.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 July 2018 - 13 July 2018

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)

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

other: epidermal keratinocytes

Cell source:

other: MatTek Corporation, Ashland MA, U.S.A.

Source strain:

other: not applicable

Vehicle:

unchanged (no vehicle)

Remarks:

The skin was moistened with 25 μL Milli-Q water.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ tissue
- Tissue batch number(s): 28831

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 minutes exposure and 60 minute exposure: 37 ± 1.0 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item.
- Observable damage in the tissue due to washing: no

TEST FOR DIRECT MTT REDUCTION AND COLOUR INTERFERENCE
KW-2200 was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, at least 25 mg of the test item or 50 μL Milli-Q water as a negative control were added to 1 mL MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. At the end of the exposure time it was checked if a blue / purple color change or a blue / purple precipitate was observed.
KW-2200 was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the color interference, at least 25 mg of the test item or 50 μL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple color change was observed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Microplate reader: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2 exposure times

EVALUATION
The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw).
ODc = ODraw – ODbl
The OD value representing 100% cell viability is the average OD of the negative controls (ODlt_u+MTT).
The %Viability for each sample and positive control is calculated as follows:
%Viability = (ODc/mean ODlt_u+MTT) * 100

PREDICTION MODEL / DECISION CRITERIA: see Table 1

ACCEPTABILITY CRITERIA:
1. The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
2. The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
3. In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be <= 30%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25.9 to 43.5 mg. The skin was moistened with 25 μL Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue.

Duration of treatment / exposure:

3 minutes and 60 minutes

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure / mean of 2 replicates

Value:

91

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

7.1

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure / mean of 2 replicates

Value:

76

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

7.4

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: 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.
- Acceptance criteria met for positive control: The mean relative tissue viability following the 1-hour exposure to the positive control was 7.4% (<15%).
- Acceptance criteria met for variability between replicate measurements: In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was < 7%, indicating that the test system functioned properly.

Mean Absorption in thein vitroSkin Corrosion Test with KW-2200

	3-minute application					1-hour application
	A (OD570)	B (OD570)	Mean (OD570)		SD	A (OD570)	B (OD570)	Mean (OD570)		SD
Negative control	1.724	1.762	1.743	±	0.027	1.814	1.715	1.765	±	0.070
KW-2200	1.599	1.576	1.587	±	0.016	1.382	1.294	1.338	±	0.062
Positive control	0.145	0.103	0.124	±	0.030	0.120	0.139	0.130	±	0.014

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.0429). Isopropanol was used to measure the background absorption.

Mean Tissue Viability in thein vitroSkin Corrosion Test with KW-2200

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
KW-2200	91	76
Positive control	7.1	7.4

Coefficient of Variation between Tissue Replicates

	3 minute	1 hour
Negative control	2.1	5.5
KW-2200	1.4	6.3
Positive control	29	14

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

Interpretation of results:

other: Not corrosive

Remarks:

According to Regulation (EC) No. 1272/2008 and its amendments.

Conclusions:

The results of an in vitro skin corrosion test showed that KW-2200 was not corrosive to the skin.

Executive summary:

The substance was tested in duplicate in an in vitro skin corrosion test according to OECD TG 431 test guideline and GLP principles. Tissues were exposed to KW-2200, a negative control (Milli-Q water) and a positive control (8.0 N KOH) for 3 minutes and 60 minutes. The substance was tested for direct MTT reduction and colour interference and both results were negative. Acceptability criteria for the negative control, positive control and variability between measurements were met.

The cell viability of the tissues exposed to KW-2200 were 91% and 76% for 3 minutes and 60 minutes exposure, respectively. Both values did not exceed thereshold for corrosivity (50% after 3 minutes exposure and 15% after 60 minutes exposure), therefore the substance is considered not to be corrosive.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 July 2018 - 17 July 2018

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)

Version / remarks:

09 October 2017

Deviations:

no

GLP compliance:

yes

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- 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.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Indication of any existing defects or lesions in ocular tissue samples: 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.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 310.1 to 369.5 mg per cornea

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

Three corneas were selected at random for each treatment group.

Details on study design:

SELECTION AND 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 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.

NEGATIVE CONTROL USED
- Amount(s) applied (volume or weight with unit): 750 μL of physiological saline per cornea

POSITIVE CONTROL USED
Amount(s) applied (volume or weight with unit): 750 μL per cornea
Concentration (if solution): 20% (w/v) Imidazole

APPLICATION DOSE AND EXPOSURE TIME
240 ± 10 minutes at 32 ± 1°C

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
- Washing: yes (with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation) and thereafter with cMEM)
- Time after start of exposure: 240 minutes

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
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 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.
- Corneal permeability:
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.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)
Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints.

DECISION CRITERIA:
A test substance that induces an IVIS ≤ 3 is not classified for eye irritancy (UN GHS: no category);
A test substance that induces an IVIS > 55 is defined as a corrosive or severe irritan (UN GHS: catgeory 1);
For a test substance that induces an IVIS >3 and ≤ 55, no prediction on irritant potency can be made.

ACCEPTABILITY CRITERIA
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Irritation parameter:

in vitro irritation score

Run / experiment:

single run

Value:

108

Negative controls validity:

valid

Remarks:

In Vitro Score 1.6

Positive controls validity:

valid

Remarks:

In Vitro Score 159

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
The corneas were clear with dark spots after the 240 minutes of treatment with KW-2200. Since the test item was solidified after 240 minutes, the test item could not be removed completely from the corneas. No pH effect of the test item was observed on the rinsing medium.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: 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.
- Acceptance criteria met for positive control: The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 159 and 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.

Summary of Opacity, Permeability and In Vitro Scores

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score^1, 2
Negative control	1.5	0.006	1.6
Positive control	123	2.387	159
KW-2200	12	6.417	108

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).

Interpretation of results:

other: Eye irritant category 1

Remarks:

According to Regulation (EC) 1272/2008 and its amendments.

Conclusions:

Based on the outcome of a Bovine Corneal Opacity and Permeability test (BCOP) performed according to OECD 437 guideline and GLP principles, it is concluded that KW-2200 induced serious eye damage.

Executive summary:

A Bovine Corneal Opacity and Permeability test (BCOP) was performed with KW-2200 according to OECD guideline 437 and GLP principles. KW-2200 was tested as it is. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 159, and the mean in vitro irritancy score of the negative control (physiological saline) was 1.6. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. KW-2200 induced serious eye damage through both endpoints, resulting in a mean in vitro irritancy score of 108 after 240 minutes of treatment.

Since KW-2200 induced an IVIS > 55, the substance should be classified as serious eye damage category 1 and labelled with H318: Causes serious eye damage, according to Regulation (EC) 1272/2008 and its amendments.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

The substance was tested in triplicate in an in vitro skin irritation test according to OECD TG 439 test guideline and GLP principles. Tissues were exposed to KW-2200, a negative control (PBS) and a positive control (5% SDS) for 15 minutes. The substance was tested for direct MTT reduction and colour interference and both results were negative. Acceptability criteria for the negative control, positive control and variability between measurements were met.

The cell viability of the tissues exposed to KW-2200 was 101% for 15 minutes exposure. Since the mean relative tissue viability for KW-2200 was above 50%, KW-2200 is considered to be non-irritant.

Skin corrosion:

The substance was tested in duplicate in an in vitro skin corrosion test according to OECD TG 431 test guideline and GLP principles. Tissues were exposed to KW-2200, a negative control (Milli-Q water) and a positive control (8.0 N KOH) for 3 minutes and 60 minutes. The substance was tested for direct MTT reduction and colour interference and both results were negative. Acceptability criteria for the negative control, positive control and variability between measurements were met.

The cell viability of the tissues exposed to KW-2200 were 91% and 76% for 3 minutes and 60 minutes exposure, respectively. Both values did not exceed thereshold for corrosivity (50% after 3 minutes exposure and 15% after 60 minutes exposure), therefore the substance is considered not to be corrosive.

Eye irritation:

A Bovine Corneal Opacity and Permeability test (BCOP) was performed with KW-2200 according to OECD guideline 437 and GLP principles. KW-2200 was tested as it is. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 159, and the mean in vitro irritancy score of the negative control (physiological saline) was 1.6. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.KW-2200 induced serious eye damage through both endpoints, resulting in a mean in vitro irritancy score of 108 after 240 minutes of treatment.

Since KW-2200 induced an IVIS > 55, the substance should be classified as serious eye damage category 1 and labelled with H318: Causes serious eye damage, according to Regulation (EC) 1272/2008 and its amendments.

Justification for classification or non-classification

Based on the available study results, KW-2200 does not have to be classified and has no obligatory labelling requirement for skin irritation/corrosion according to Regulation (EC) No 1272/2008 and its amendments.

KW-2200 needs to be classified as serious eye damage category 1 and labelled with H318: Causes serious eye damage, according to Regulation (EC) 1272/2008 and its amendments.