Pentyl D-glucoside

Endpoint summary

Description of key information

Skin irritation / corrosion in vitro (In Vitro Skin Irritation: Reconstructed Human Epidermis Test, OECD 439): not irritating

Eye irritation / serious eye damage (WoE, Bovine Corneal Opacity and Permeability Test, OECD 437; Reconstructed Human Cornea-like Epithelium (RhCE) Test, OECD 492): irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 - 14 Nov 2016

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:

23 July 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM, Medicines & Healthcare products Regulatory Agency

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: SkinEthic Laboratories, Lyon, France

Source strain:

other: EPISKIN™; reconstructed three-dimensional human epidermis

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST METHOD:
The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN reconstructed human epidermis model after a treatment period of 15 min followed by a post-exposure incubation period of 42 h. The EPISKIN model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt within the mitochondria of viable cells in the test item treated tissues relative to the negative controls.

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE:
- Model used: EPISKIN™ Reconstructed Human Epidermis Model
- Tissue batch number: 16-EKIN-045
- Delivery date: 08 November 2016
- Maintenance Medium lot number: 16-MAIN3-075
- Assay Medium lot number: 16-ESSC-048

ADAPTATION TO CELL CULTURE CONDITIONS (PRE-INCUBATION):
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item (positive and negative). The tissues were incubated at 37 °C, 5% CO2 in air overnight.

TREATMENT AND RINSING:
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with 10 µL of the test item. The test item was applied topically to the corresponding tissues ensuring uniform covering. Triplicate tissues treated with 10 μL of Dulbecco’s Phosphate Buffered Saline (DPBS) served as the negative controls and triplicate tissues treated with 10 μL of Sodium Dodecyl Sulphate (SDS) 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item, the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). The plates were kept at room temperature for 15 min.

At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by means of a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 h.

MTT LOADING, FORMAZAN EXTRACTION AND OPTICAL DENSITY MEASUREMENT.
Following the 42 h post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 min to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination.
2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 h at 37 °C, 5% CO2 in air. At the end of the 3 h incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C for 72 h, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution. For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 562 nm (without a reference filter) using the Anthos 2001 microplate reader.

NUMBER OF REPLICATE TISSUES: triplicates

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL:
- The test substance is considered to be irritating to skin if the viability is less than or equal to 50% after a 15 min exposure period followed by a 42 h post-exposure incubation period
- The test substance is considered to be not irritating to skin if the viability is greater than 50% after a 15 min exposure period followed by a 42 h post-exposure incubation period

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL (26.3 μL/cm2)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5% (w/v)

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

The test was performed in triplicates for each test or control group.

Species:

other: in vitro system

Type of coverage:

other: in vitro system

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean of all 3 test item tissues

Value:

90.4

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:

OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no, as assessed in a suitable pre-experiment
- Colour interference with MTT: no, as assessed in a suitable pre-experiment
- IL-1alpha analysis: considered unnecessary as the results of the MTT test were unequivocal

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, OD of negative control 0.836 (criterion: OD ≥ 0.8 and ≤ 2.8)
- Acceptance criteria met for positive control: yes, tissue viability 11.8% (criterion: tissue viability ≤ 20%)
- Acceptance criteria met for variability between replicate measurements: yes, SD 1.3% (negative control), 4.2% (positive control), 8.3% (test item) (criterion: ≤ 18%)

Table 1: Mean OD₅₆₂Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD562 of tissues	Mean OD562 of triplicate tissues	± SD of OD562	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative control item	0.839	0.836	0.011	100.4	100*	1.3
	0.846			101.2
	0.824			98.6
Positive control item	0.138	0.099	0.035	16.5	11.8	4.2
	0.089			10.6
	0.070			8.4
Test item	0.752	0.756	0.070	90.0	90.4	8.3
	0.827			98.9
	0.688			82.3

OD = Optical Density

SD = Standard deviation

∗= The mean viability of the negative control tissues is set at 100%

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.

Conclusions:

There is regulatory acceptance in the EU that a substance can be considered to be not irritating to skin based on an appropriate result in the human epidermis model test (OECD 439).

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

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

03 Feb 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:

26 July 2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM, Medicines & Healthcare products Regulatory Agency

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: local abattoir
- Characteristics of donor animals: adult animals (typically 12 to 60 months old)
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Isolated eyes were stored in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter.
- Time interval prior to initiating testing: The corneas were refrigerated on arrival and used within 24 hours of receipt.
- indication of any existing defects or lesions in ocular tissue samples: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
- Indication of any antibiotics used: penicillin at 100 IU/mL and streptomycin at 100 μg/mL

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.75 mL

NEGATIVE CONTROL
- Amount(s) applied: 0.75 mL

POSITIVE CONTROL
- Amount(s) applied: 0.75 mL
- Purity: > 99.8%

Duration of treatment / exposure:

10 min at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

120 min at 32 ± 1 °C

Number of animals or in vitro replicates:

triplicates for each treatment and control groups

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing Hanks’ Balanced Salt Solution (HBSS) until they were mounted in special BCOP holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes.

QUALITY CHECK OF THE ISOLATED CORNEAS
At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated. Three corneas with opacity values close to the median value of all corneas were allocated to the negative and positive control as well as to the test item.

APPLICATION DOSE AND EXPOSURE TIME
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 4
- POST-EXPOSURE INCUBATION: Test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of Anthos 2001 microplate reader (OD492)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) :
In Vitro Irritancy Score (IVIS) = mean opacity value + (15 x mean permeability OD492 value)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as severe irritant/corrosive and labelled Category 1.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; ≤ 55: no prediction can be made.

Irritation parameter:

in vitro irritation score

Value:

21.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
The corneas treated with the test item were cloudy post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, opacity = 2.7, permeability = 0.030 (criterion: opacity ≤ 2.8 and permeability ≤ 0.115)
- Acceptance criteria met for positive control: yes, IVIS = 43.3 (criterion: IVIS within two standard deviations of the historical mean collated during 2015 for this testing facility [IVIS 27.2 to 53.4])

 

Table 1: Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea number	Opacity					Permeability (OD)		In Vitro Irritancy Score (IVIS)
		Pre-Treatment	Post-Treatment	Post-Incubation	Post-Incubation – Post-Treatment	Corrected value		Corrected value
Negative control	10	3	4	6	3		0.052
	12	4	5	6	2		0.016
	14	4	4	7	3		0.021
					2.7*		0.030**		3.1
Positive control	11	2	25	29	27	24.3	1.336	1.306
	13	5	28	31	26	23.3	1.319	1.289
	15	2	31	30	28	25.3	1.256	1.226
						24.3***		1.274***	43.4
Test item	16	5	16	22	17	14.3	0.798	0.768
	17	5	16	21	16	13.3	0.418	0.388
	18	3	14	18	15	12.3	0.480	0.450
						13.3***		0.536***	21.4

 

OD = Optical density

*: Mean of the post-incubation – pre-treatment values

**: Mean permeability

***: Mean corrected value

 

Interpretation of results:

other: no prediction of eye irritation can be made

Conclusions:

Under the conditions of this in vitro Bovine Corneal Opacity and Permeability Test (BCOP), the test item caused an increase of the corneal opacity and of the permeability above the values characteristic for substances not requiring classification as eye irritants according to the criteria of Regulation (EC) No. 1272/2006 (CLP). The calculated mean in vitro irritancy score (IVIS) was 21.4. However, the values determined are not sufficiently high to indicate the test item to cause serious eye damage. In conclusion, no prediction of the eye irritation potential of the test item can be made and further data are required.

The test item was shown to have an irritating or corrosive potential towards reconstructed human epidermis tissue. Moreover, in a Bovine Corneal Opacity and Permeability Test capable of identifying substances that can induce serious eye damage and substances not requiring classification for eye irritation or serious eye damage, no prediction could be made for the registered substance. In conclusion, accounting for both in vitro investigations in a Weight-of-Evidence approach, the registered substance is considered to meet the criteria for classification as Eye Irrit. 2, H319, according to the CLP Regulation (EC) No. 1272/2008.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

21 Sept - 12 Oct 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:

July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Mainzer Straße 80, D-65189 Wiesbaden

Species:

human

Strain:

other: EpiOcular™

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The MatTek EpiOcular™ model has been widely used as a research and testing model for many years and for a great variety of chemicals.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 µL
- Concentration: 70% (in water)

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL

Duration of treatment / exposure:

30 mins at 37 ± 1.5 °C, 5 ± 0.5% CO2, 95% relative humidity

Duration of post- treatment incubation (in vitro):

post-soak immersion: 12 min
post-treatment incubation: 120 min at 37 ± 1.5 °C in humidified atmosphere of 5 ± 0.5% CO2

Number of animals or in vitro replicates:

The test was performed on a total of 2 tissues per dose group.

Details on study design:

- Details of the test procedure used
- EpiOcular™ supplier: MatTek Corporation, 82105 Bratislava, Slovakia
- Lot No. of EpiOcular™ kit: 27008

ASSESSMENT OF DIRECT MTT REDUCTION BY THE TEST ITEM
Approximately 50 μL of the test item were added to a 1 mL of a 1.0 mg/mL MTT solution (in Dulbecco's Minimum Essential Medium, DMEM) in a glass tube and the mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for 3 h. A control (50 μL of deionised water in 1 mL of 1.0 mg/mL MTT solution) was performed concurrently. The MTT solution colour did not turn blue/purple. The test item is not presumed to be a MTT reducer. An additional test with freeze-killed tissues did not have to be performed.

ASSESSMENT OF COLOURED OR STAINING MATERIALS
Since the test item was non-coloured, additional tests had to be performed to assess if it becomes coloured after contact with water or isopropanol. For this purpose 50 μL of the test item were added to 1.0 mL of water and to 2 mL isopropanol in a glass tube. The water mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for 1 h, the isopropanol mixture for 3 h at room temperature. The test item did not dye water or isopropanol. Additional tests with viable tissues did not have to be performed.

EXPERIMENTAL PROCEDURE
EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 6-well plates. Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for 1 h in the Assay Medium. After 1 h, the Assay Medium was replaced by 1 mL fresh Assay Medium at 37 °C and the EpiOcular™ tissues were incubated at standard culture conditions overnight (about 16 h).
After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca++ and Mg++ free Dulbecco's Phosphate Buffered Saline (DPBS). The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2, 95% relative humidity) for 30 min. After the 30 min Ca++ Mg++ free DPBS pre-treatment, the test and control items were tested by applying 50 μL topically on the EpiOcular™ tissues. The tissues were incubated at standard culture conditions for 30 min. At the end of the 30 min treatment time, the test item was removed by extensively rinsing the tissues with Ca++ Mg++ free DPBS.
After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for a 12 min immersion incubation (post-soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue. At the end of the post-soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the inserts were blotted on absorbent material, and transferred to the appropriate well of the pre-labelled microtiter plate containing 1 mL of warm Assay Medium. The tissues were incubated for 120 min at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 min at standard culture conditions. Each insert was removed from the 24-well plate after 180 min, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plates were sealed with a standard plate sealer and were stored overnight at 2 - 8 °C in the dark and then shaken for 2 - 3 h at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken. The extract solution was mixed and two 200 μL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.
The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1). No reference wavelength measurement was used.

DATA EVALUATION
1) The mean optical density (OD) value of the blank control wells (ODBlk) for each experiment was calculated.
2) The mean value of the two replicates for each tissue was calculated.
3) The mean ODBlk from each mean OD value of the same experiment was subtracted (blank corrected values).
4) The mean value of the two relating tissues for each control (negative control (NC) and positive control (PC) and test item (TI) was calculated (ODTI, ODNC, ODPC).
5) The mean OD value of the negative control corresponds to 100% viability. Corrected negative control OD = Negative Control OD - ODBlk = 100% Viability

CALCULATION OF THE VIABILITY
1) The percent viability of each of the two relating tissues for each control and test item relative to the negative control (= 100%) was calculated.

Viability (%) = 100 x (ODTi / ODPC / ODNC) / mean ODNC

2) The difference of the viability between duplicate tissues was calculated. If the difference is > 20% the test is considered as non-qualified.
3) The mean test item viability (TI viability) was calculated and the test item was classified according to the prediction model.

PREDICTION MODEL
If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labeled non-irritant (no Category according to UN GHS). If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labeled irritant (Category 2 or Category 1 according to UN GHS; no differentiation between the categories possible).

ACCEPTANCE CRITERIA
The results are acceptable according to MatTek Protocol, if:
- mean absolute OD570 nm of the negative control is > 0.8 and < 2.5
- mean relative tissue viability of the positive control is < 50%
- relative tissue viability difference of replicate tissues is < 20%.

Irritation parameter:

other: mean relative viability (%)

Run / experiment:

30 min

Value:

6.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue/purple colour.
- Colour interference with MTT: The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol did not lead to a change in colour.
- Effect of test item: The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 6.7% (threshold for irritancy ≤ 60%), consequently the test item was irritant to eye.

ACCEPTANCE OF RESULTS:
- The negative control OD is > 0.8 and < 2.5 (1.610 and 1.674).
- The mean relative viability of the positive control is below 50% of the negative control viability (34.1%).
- The difference of viability between the two relating tissues of a single item is < 20% (values between 0.9% and 1.7%) in the same run (for positive and negative control tissues and tissues of single test items).

Table 1: Results after treatment for 30 min with test item and controls

Treatment Group	Tissue No.	OD 570 nm Well 1	OD 570 nm Well 2	Mean OD of 2 Wells	Mean OD of 2 Wells blank corrected	Mean OD of Treatment Group blank corrected	Rel. Viability [%] Tissue	Absolute Value of the Difference of Rel. Viability  Tissue [%]	Mean Rel. Viability [%]
Blank		0.035	0.036	0.035
Negative Control	1	1.674	1.636	1.655	1.619	1.606	100.8	1.7	100.0
	2	1.610	1.645	1.628	1.592		99.2
Positive Control	1	0.592	0.588	0.590	0.554	0.547	34.5	0.9	34.1
	2	0.577	0.574	0.576	0.540		33.6
Test Item	1	0.133	0.133	0.133	0.097	0.108	6.1	1.4	6.7
	2	0.154	0.155	0.155	0.119		7.4

Table 2: Historical data

Positive Control		Negative Control [OD570]
Mean Viability	32.3%	Mean Absorption	1.65
Rel. Standard Deviation	11.1%	Rel. Standard Deviation	0.295
Range of Viabilities	6.90% - 43.4%	Range of Absorbance	1.27 – 2.16
Mean Absorption	0.566
Rel. Standard Deviation	0.283
Range of Absorbance	0.107- 0.943

Data of 30 studies performed from July 2015 until middle of November 2016

Interpretation of results:

other: irritating potential (Eye Irrit. 2 or Eye Dam. 1 according to Regulation (EC) No. 1272/2008)

Conclusions:

Under the conditions of the test, the test item was shown to have an irritating or corrosive potential towards reconstructed human epidermis tissue in the EpiOcular™ EIT prediction model. The result does not allow for the classification of the registered substance as irritant or corrosive and further data are required.

In a Bovine Corneal Opacity and Permeability Test capable of identifying substances that can induce serious eye damage and substances not requiring classification for eye irritation or serious eye damage, no prediction could be made for the registered substance. Moreover, the registered substance was shown to have an irritating or corrosive potential towards reconstructed human epidermis tissue. In conclusion, accounting for both in vitro investigations in a Weight-of-Evidence approach, the registered substance is considered to meet the criteria for classification as Eye Irrit. 2, H319, according to the CLP Regulation (EC) No. 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The skin irritation / corrosion potential of D-Glucopyranose, oligomeric, pentyl glycosides (CAS 100231-63-8) was investigated in an in vitro skin irritation test using reconstructed human epidermis. No skin irritation potential was identified.

Based on an in vitro study with reconstructed human cornea-like epithelium (RhCE) and on a bovine corneal opacity and permeability test, D-Glucopyranose, oligomeric, pentyl glycosides (CAS 100231-63-8) was identified as irritating to eyes.

Justification for classification or non-classification

The available data on skin irritation for D-Glucopyranose, oligomeric, pentyl glycosides (CAS 100231-63-8) do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 (CLP). Data are, therefore, conclusive but not sufficient for classification. The available data on eye irritation / serious eye damage, however, meet the criteria for classification as Eye Irrit. 2, H319, according to the CLP regulation.