D-Glucitol, 1-deoxy-1-[methyl(1-oxononyl)amino]-

Endpoint summary

Description of key information

To assess skin irritation/corrosion potential of the test itam following studies were performed in an bottom-up aapproach:

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

2) In vitro skin corrosion: reconstructed human epidermis (RHE) test method (OECD 431)

To determine the potential for ocular irritation of the test item the bovine corneal opacity and premeability (BCOP) was performed according to OECD 437.

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:

23-02-2017 to 12-05-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Storage: room temperature
Description: White powder
Expiry date: 2017-12

Test system:

human skin model

Source species:

human

Vehicle:

water

Details on test system:

This study is designed based on MatTek protocol "in vitro EpidDerm skin corrosion test". EpiDerm TM tissue, Lot 25700 Kit P, were received from MatTek Corporation (Ashland, MA) on 28 Feb 2017 and refrigerated at 2-8 °C. Before use, tissue were incubated (37°C +/- 1 °C, 5% +/- 1% CO2) with assay medium (Mat Tek) for a one-hour equilibration. Equilibration medium was replaced with fresh medium before dosing.
25 mg of the test item, plus 25 µl H2O were applied to the top of each EpiDerm tissue. The test article remained in contact with the EpiDerm tissue for 3 and 60 minutes. A negative control (H2O) and a positive control (50 µl of KOH) were tested in the same manner (all treatments were conducted in duplicates).

At the end of the exposure period, each EpiDerm tissue was rinsed with PBS and transferd to a 24-well microplate containing 300 µl MTT solution (1 mg/ml MTT in DMEM). The tissues were then returned to the incubator for a 3-hour MTT incubation period. Following the MTT incubation period, each Epiderm tissue was rinsed with PBS and then treated overnight with 2.0 ml of extractant solution (isopropanol) per well. The absorbency of an aliquot of the extracted MTT formazan was measured in triplicate at 540 nm usin a microplate reader (µQuant Plate Reader, bio-Tek Instruments, Winooski, VT).

The direct reduction of MTT by the test item was investigated in advance and revealed that the test item did not reduce MTT.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg of the test Item, plus 25 µl of TCh2O, were applied to the top of each EpiDerm tissue.

Duration of treatment / exposure:

3 and 60 minutes

Number of replicates:

each group in duplicate (test item positive and negative control)

Irritation / corrosion parameter:

% tissue viability

Remarks:

3 min

Run / experiment:

mean (Test item)

Value:

90.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

60 min

Run / experiment:

mean (test item)

Value:

30.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

3 min

Run / experiment:

mean (negative control)

Value:

100

Vehicle controls validity:

valid

Remarks:

Water

Negative controls validity:

valid

Remarks:

Water

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

60 min

Run / experiment:

mean (negative control)

Value:

100

Vehicle controls validity:

valid

Remarks:

Water

Negative controls validity:

valid

Remarks:

Water

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

3 min

Run / experiment:

mean (positive control)

Value:

31

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Potassium Hydroxide Solution

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean (positive control)

Value:

1.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Potassium Hydroxide Solution

Remarks on result:

positive indication of irritation

Test and Control article	Mean Viability (3 min)	Mean viability (60 min)	Predicted corrosivity
test item	90.2%	30.1%	Non-Corrosive
Tissue culture water (negative control)	100.0%	100.0%	Non-Corrosive
Potassium hydroxide Solution, 8.0N (positive control)	31.0%	1.7%	Corrosive

Interpretation of results:

GHS criteria not met

Conclusions:

non-corrosive

Executive summary:

To predict the skin corrosivity potential of the test item In vitro skin corrosion: reconstructed human epidermis (RHE) test method was used according to OECD test guidline 431. This study is designed based on MatTek protocol "in vitro EpidDerm skin corrosion test". 25 mg of the test item, plus 25 µl H2O were applied to the top of each EpiDerm tissue. The test article remained in contact with the EpiDerm tissue for 3 and 60 minutes. A negative control (H2O) and a positive control (50 µl of KOH) were tested in the same manner (all treatments were conducted in duplicates). The viability of the tiseues after the incubation period were measured by using MTT. The light absorption of the extracted MTT from each sample was measured at 54 nm using a microplate reader. Th emean absorbance value for each time point was calculated from the optical density (OD) of the duplicate tissues and expressed as precent viability for each sample using following formula:

% viability= 100 x (ODsample/OD negative)

Based on following results and according the prediction model for EpiDerm (OECD TG 431) the test item is considered to be non-corrosive.

Test and Control Article Identity	Mean viability (3 min.)	Mean viability (60 min.)	Predicted Corrosivity
Test Item	90.2 %	30.1 %	Non-Corrosive
Negative control (H2O)	100 %	100 %	Non-Corrosive
Positive Control (KOH)	31.0 %	1.7 %	Corrosive

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13-04-2017 to 18 -07-2017

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:

July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Storage: room temperature
Description: White powder
Expiry date: 2017-12

Test system:

human skin model

Justification for test system used:

Recommended in vitro model

Details on test system:

Test System: Epidermal Model, MatTek EpiDerm™ (EPI-200-SIT) kit.
Justification: The EpiDerm Skin Model closely parallels human skin, thus providing a useful in vitro means to assess dermal irritancy and toxicology.
Storage: MatTek EpiDerm™ kit components and assay medium will be stored at 2-8ºC.
EXPERIMENTAL DESIGN:
Reduction of Methyl thiazole tetrazolium (MTT): MTT 1 mg/ml, diluted in Dulbecco's Modified Eagle's Medium (DMEM) will be prepared and protected from light. MTT reduction by the test article will be measured prior to dosing, unless the test article is not suitable for this testing. If the test article results in a reduction of MTT or is likely to stain the tissue, frozen (dead) tissues will be run concurrently for each time point tested (or at minimum, the longest time point tested).
Pre-Incubate: Upon receipt, EpiDerm™ tissues will be placed in 6-well plates containing prewarmed room temperature assay media and will be equilibrated in a humidified 37ºC ± 1ºC, 5% ± 1% CO2 incubator for at least one hour. After the first one hour pre-incubation, the tissues will be moved to new wells with fresh medium, and a second incubation will be conducted overnight (18 ± 3hr) at 37ºC ± 1ºC, 5% ± 1% CO2.

Dose Groups
1. Negative control 30 µL PBS
2. Positive control 30 µL 5% SDS solution
3. Test Item 25 mg
The test was performed on a total of 3 tissues per dose group.
SDS sodium dodecyl sulfate
PBS Dulbecco's phosphate buffered saline

EpiDerm™ tissues will be topically exposed to the test article and control articles for 60 minutes, in triplicate. Tissues will be returned to the incubator for 35 ± 1 minutes, then returned to the sterile hood for the remainder of the 60-minute exposure period. Rinse: After dosing and incubation, the tissues will be thoroughly rinsed with PBS, blotted to remove the test substance and dry the tissue, and transferred to fresh medium.
MTT Conversion: Following the 42-hour incubation, each insert will be individually rinsed with PBS and the tissue will be incubated with 300 μl of 1 mg/ml MTT, diluted in Dulbecco's Modified Eagle's Medium (DMEM), for three hours at 37ºC ± 1oC, 5% ± 1% CO2. MTT solution is prepared the day of use and protected from light.
MTT Extraction: Following the MTT incubation period, each insert will be removed individually and gently rinsed with PBS to remove any residual MTT solution. The inserts will be immersed using 2.0 ml of isopropanol extractant solution per well, completely covering the EpiDerm™ tissue. The extraction plate will be sealed and covered to reduce evaporation of extractant.
Extraction Conditions: The extraction will be allowed to proceed for at least two hours, with shaking, at room temperature. Alternatively, the extraction can be allowed to proceed overnight at room temperature in the dark.
Measuring Optical Density: The optical density of the extracted samples will be determined at a single wavelength of 540 nm, using 200 μl of the extractant as a blank.
Calculating Percent Viability: The percent relative cell viability will be determined for each tissue using the following formula:
% viability = 100 x [OD(sample)/OD(negative control)]

Control samples:

yes, concurrent negative control

yes, concurrent vehicle

Amount/concentration applied:

The EpiDerm™ tissue will be moistened with 25 μl PBS before dosing. 25 mg of powder or solid material (ground into fine powder) will be added into the Millicell atop the tissue. Fibrous or cloth-like test articles will be cut to the appropriate dimensions to cover the tissue insert. For test articles that cannot be pipetted, applicator pins inverted onto the tissue
will be used to provide a reproducible, even means of application.

Duration of treatment / exposure:

EpiDerm™ tissues will be topically exposed to the test article and control
articles for 60 minutes, in triplicate.

Duration of post-treatment incubation (if applicable):

The six-well plates containing the rinsed EpiDerm™ tissues will be returned to the incubator for 24 ± 2 hours. Medium will be changed at 24 ± 2 hrs. Tissues will be returned to incubator for an additional 18 ± 2 hours.

Number of replicates:

3 per group

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean (test item)

Value:

3.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean (positive control)

Value:

3.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean (negative control)

Value:

100

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

test and control article	Mean tissue viability	irritancy classification
test item	3.4%	Irritant
Phosphate buffered Saline (negative control)	100 %	Non-irritant
5% Sodium dodecyl sulfate (positive control)	3.3 %	Irritant

Interpretation of results:

other:

Executive summary:

To predict dermal irritation potential of test item the In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method. according to OECD TG 439 was performed. This study is designed based on MatTek protocol in vitro EpiDermTM Skin Irritation Test.

MatTek EpiDerm™ tissue samples were treated in triplicate with the test article,

negative control and positive control for 60 minutes. Following treatment and subsequent incubation

time, the viability of the tissues was determined using methyl thiazole tetrazolium (MTT) uptake and

reduction. The absorbance of each sample was measured at 540 nm. The viability was then expressed

as a percent of control values.

The viability was then expressed

as a percent of control values. If the mean tissue viability was ≤50%, the test material was classified as

an irritant; if the mean tissue viability was >50%, the test material was classified as a non-irritant.

The mean tissue viability of the skin after treatemet with the test item was 3.4% (≤50%) . Based on these result the test item was classified as an skin irritant.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24-02-2017 to 12-05-2017

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)

Version / remarks:

Adopted 26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: Bovine corneas

Strain:

not specified

Details on test animals or tissues and environmental conditions:

The bovine eyes, in a Hanks' Balance Salt Soluation (HBSS) with penicillin-streptomycin, were received from Spear Products on 02 March 2017 and transported to the testing facility in a refrigerated container. The eyes were examined prior to use on the day of dosing. Any eye with a cornea exhibiting evidance of vascularization, pigmentation, opacity or scratches was discarded. The dissected corneas were mounted in specially designed holders that were seperated into anterior and posterior chambers and filled separately. Ech cornea was mounted allowing the epithelium of the cornea to project into the anterior chamber. The posterior chamber was filled with MEM solution ensuring contact with the endothelium. The anterior chamber was filles MEM solution, ensuring contact with the epitheliu,. Each cornea was visually inspected again to ensure there were no defects. The entire holder was incubated at 32 (+/- 1)°C and allowed to equilibrate for at least one hoer, but nozt longer than two hours.

Vehicle:

physiological saline

Amount / concentration applied:

0.75 ml of the 20 % (w(v) test article formulation in saline

Duration of treatment / exposure:

After 4 hours ± 10 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red).

Observation period (in vivo):

1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C.

Duration of post- treatment incubation (in vitro):

4 hours (+/- 10 min) exposure with the test item

Number of animals or in vitro replicates:

3 corneas for the test item:
Thereof: 3 corneas for the test item for opacity measurement, 3 corneas for the test item for permeability measurement
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive controls treated with imidazolen in physiological saline 0.9% NaCl

Details on study design:

Preparation of the Corneas:
The bovine eyes (at least six months old), in a Hanks' Balance Salt Soluation (HBSS) with penicillin-streptomycin, were received from Spear Products on 02 March 2017 and transported to the testing facility in a refrigerated container. The eyes were examined prior to use on the day of dosing. Any eye with a cornea exhibiting evidance of vascularization, pigmentation, opacity or scratches was discarded. The dissected corneas were mounted in specially designed holders that were seperated into anterior and posterior chambers and filled separately. Ech cornea was mounted allowing the epithelium of the cornea to project into the anterior chamber. The posterior chamber was filled with MEM solution ensuring contact with the endothelium. The anterior chamber was filles MEM solution, ensuring contact with the epithelium. Each cornea was visually inspected again to ensure there were no defects. The entire holder was incubated at 32 (+/- 1)°C and allowed to equilibrate for at least one hoer, but not longer than two hours. Following the equilibration, the MEM solution will be removed from both chambers and the chambes refilled with fresh MEM.
A pre-exposure determination of opacity will be made for each designated cornea following the equilibration period by measuring each against the blank supplied with the OP-KIT opacitometer. Any cornea with a value > 7 units will be discarded.
A 20% (200 mg/ml) solution or susspension of the solid test article in 0.9% Saline will be prepared. A volume of 0.75 ml of the dosing solution, MEM solution or imidazole solution will be applied to the epithelium in the anterior chamber in a manner that ensures that entire cornea will be covered. All corneas will be returned to the 32 (+/-1) °C incubator for 4 hours ( +/- 10 min) in a horizontal position. Following the 4-hour exposure, the test article solution, MEM or Imidazole solution will be removed from the epithelium of cornea and the anterior chamber by washing with MEM solution containing phenol red. A final rinse will be made with MEM without phenol red. Both ethe anterior and posterior chambers will be refilled with fresh MEM solution and a measurment of opacity obtained with each treated cornea compared to the blank supplied with the OP-KIT opacitometer.

Differences in light transmission through the control and treated cornea willbe determined using an OP-KIT opacitometer produced by Electro-Design Corporation of Riom, France. Each treated cornea will be scored in comparison to the blank provided with the OP-KIT opacitometer.

The In vitro IVIS Scre (IVIS) will be calculated b yadding the corrected mean opacity score to 15 times the corrected mean optical density. The calculations to obtain an IVIS for the positive control will be performed in the same manner as the test item
IVIS= Corrected mean Opacity score + (15 x Corrected Mean Optical Density score)
The IVIS for the negative control will be calculated by adding the mean opacity score to 15 times the mean optical density.
IVIS= Mean Opacity Score + ( 15 x Mean Optical Density score)

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean value test item

Value:

66.31

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

mean test Item

Value:

43

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

Positive control (Imidazole)

Value:

84.15

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

Mean positive control

Value:

68.34

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

Men vehicle control (0.9% Sodium Chloride Irrigation)

Value:

1.48

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

Mean Vehicle control (0.9% Sodium Chloride Irrigation)

Value:

1.67

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean negative control (MEM)

Value:

1.72

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

Mean Negative control (MEM)

Value:

1.33

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Based on an IVIS greater than 55 (IVIS= 66.31), and as defined in OECD Guidline No 437, the tets article is in UN GHS Category 1 and is considered to be a severe/ corrosive eye irritant.

Executive summary:

To determine the potential for ocular irritation of the test item the bovine corneal opacity and premeability (BCOP) was performed according to OECD 437. The bovine Corneas per group were dosed with 0.75 ml of a 20% (200mg/ml) formulation of the test item in 0.9% Sodium chloride Irrigation (saline), Minimal Essential Media (MEM, negative contl), or a 20 % formulation of imidazole in 0.9 % saline (positive control). Vehicle control were dosed with 0.75 ml of 0.9% Sodim chloride irrigation. Following a 4-hour exposure for ech group of dosed corneas, opacity measurements and sodium fluorescin permeability were determined. Based on an IVIS greater than 55 (IVIS= 66.31), and as defined in OECD Guidline No 437, the tets article is in UN GHS Category 1 and is considered to be a severe/ corrosive eye irritant.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

To assess skin irritation/corrosion potential of the test itam following studies were performed in an bottom-up aapproach:

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

2) In vitro skin corrosion: reconstructed human epidermis (RHE) test method (OECD 431)

Based on a positive Skin-Irritation test and a negative skin-corrosion test the test item is considered to be skin irritating.

To determine the potential for ocular irritation of the test item the bovine corneal opacity and premeability (BCOP) was performed according to OECD 437. Based on an IVIS greater than 55 (IVIS= 66.31), and as defined in OECD Guidline No 437, the tets article is in UN GHS Category 1 and is considered to be a severe/ corrosive eye irritant.