D-Glucose, reaction products with alcohols C16-18 (even numbered)

Endpoint summary

Description of key information

- Skin irritation: not irritating

OECD TG 439 / EU method B.46; RL1; GLP: relative mean tissue viability after 15 minutes treatment, 42 h observation: 102%.

 

- Eye irritation: irritating
OECD TG 437 (BCOP);RL1; GLP: calculated mean in vitro irritation score: 9.11; no final prediction possible
OECD TG 492 (EpiOcular),RL1; GLP: mean relative tissue viability (% negative control) 43.4% (≤ 60%)

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:

2016-12-28 to 2017-02-23

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 28, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 06, 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC\/AM DB-ALM Protocol No. 131 “EpiSkin Skin irritation Test™15 min - 42 hours.

Version / remarks:

June 09, 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This test uses the EPISKIN-SM™ reconstructed human epidermis model (SkinEthic) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

water

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
EpiSkin™ Kit Lot No.: 17-EKIN-006, SkinEthic Laboratories (69007 Lyon, France)
This skin model consists of normal human epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Date of initiation of testing: On 2017-02-07 (receipt of EpiSkin™) the tissues were transferred to 12-well plates containing 2 mL maintenance medium per well and incubated at 37°C ± 1°C, 5.0% CO2 for at least 24 h.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C ± 1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
- washed once after the 15 min incubation step with DPBS. Excess DPBS was removed by blotting bottom with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT stock solution: 3 mg/mL MTT (Sigma; Lot No.: MKBR6576V) in PBS (Gibco; Lot No.: 1813255); MTT medium: MTT stock solution was diluted 1 + 9 with DMEM-based medium (final concentration 0.3 mg/mL)
- Incubation time: 3 h
- Wavelength: 570 nm ± 30 nm
- Filter bandwidth: 30 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Barrier function: IC 50 determination (SDS concentration, MTT test, n = 14): Specification ≥ 1.5 mg/mL; Result: 2.1 mg/mL
- Morphology: Histology scoring (HES stained vertical paraffin sections, n = 6): Specification ≥ 19.5; Result: 22.8 ± 0.3, CV = 1.2%
Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
- Contamination: On blood of the same donors, the following was verified:
- the absence of HIV] and 2 antibodies
- the absence of hepatitis C antibodies
- the absence of hepatitis B antigen HBs
On epidermal cells of the same donors, the absence of bacteria, fungus and mycoplasma was verified.

NUMBER OF REPLICATE TISSUES: 3 tissues per dose group

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues were used in order to determine the non-specific reduction of MTT and if a non-specific colouring occurs that is calculated to be > 5% as compared to the negative control. The second step is necessary in order to avoid a possible double-correction for colour interference for test items which act as non-specific MTT-reducers and show non-specific colouring of living tissues.
- N. of replicates :
Three killed tissues were incubated either with 10 mg of the test item and with the negative control (DPBS; KU), respectively, and the reduction was measured in repeat determinations.
- Method of calculation used:
If the mixture of living tissues treated with the test item and the MTT-incubation medium turns blue/purple the non-specific reduction of MTT (NSMTT) is calculated using the following formula:
NSMTT [%] = [(ODKT- QDKU)/ODNK] * 100
With KT = treated tissues; KU = negative control and NK = negative control of the living tissues.
If non-specific MTT reduction was ≤ 30% relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) of the test item treated living tissues (TM) was corrected according to the following formula:
TODTT = ODTM - (ODKT - ODKU)
The non-specific colour of additional viable tissues (NSCliving) was then calculated according to the following formula:
NSCliving [%] = [ODTVT/ODUVT]*1OO
If NSCliving was ≤ 5% relative to the negative control of living epidermis, no correction of the results was necessary.
If NSCliving was > 5% and ≤ 30% relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) was corrected according to the following formula:
TODTT = QDTM — QDTVT

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test item is considered to be irritant to skin in accordance with regulation EC 1272/2008 and UN GHS “Category 2”, if the tissue viability after 15 min of exposure and 42 h of post-incubation is less or equal to 50%.
- The test substance may be considered as non-irritant to skin in accordance with UN GHS “No Category” if the tissue viability after exposure and post-treatment incubation is higher than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 ± 2 mg (26.3 mg/cm²)

VEHICLE
- Amount(s) applied (volume or weight with unit): 5µL aqua dest.

NEGATIVE CONTROL
- Concentration (if solution): DPBS (Lot No.: 1737107)

POSITIVE CONTROL
- Concentration (if solution): 5% sodium dodecyl sulfate (SDS; Lot No.: 40015277) in aqua dest.

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

three tissues were used and the viability measured in repeat determinations

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

test item

Value:

106.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

mean OD570 = 0.644

Positive controls validity:

valid

Remarks:

mean tissue viability = 15.5%

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: Yes, ≤ 5%


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline:
OD 570 ± 30 nm blank: mean: 0.043; SD: 0.001; n: 67
Absolute OD 570 ± 30 nm NK: mean: 0.866; SD: 0.120; n: 66
Relative Viability [%] PC: mean: 11.73; SD: 8.17; n: 67
SD of Viability [%]: mean: 6.43; SD: 4.68; n: 254

Interpretation of results:

GHS criteria not met

Conclusions:

D-Glucose reaction products with alcohols C16-C18 was not irritating in the in vitro skin irritation test under the experimental conditions described in this report.

Executive summary:

In a dermal irritation study performed in accordance with OECD Guideline 439 (In Vitro Skin Irritation) (adopted July 28, 2015), D-Glucose reaction products with alcohols C16-C18 was applied to the three-dimensional human epidermis model tissue for an exposure period of 15 minutes in triplicates. 5 μL of deionised water were topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. Each approximately 10 mg of the test item were applied to the wetted tissues. The test item was spread to match the surface of the tissue.

After 15 minutes exposure at room temperature, the tissues were washed with DPBS to remove any residual test material. Subsequently the tissue constructs were incubated for 42 h at 37 ± 1°C. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive (5% SDS) and negative (aqua dest.) control gave responses that were within the acceptance criteria and as such demonstrated the validity of the study.

The relative mean tissue viability obtained after 15 minutes treatment and 42 h post-incubation with D-Glucose reaction products with alcohols C16-C18 compared to the negative control tissues was 102 %. Since the mean relative tissue viability for the test substance was above 50%, the test item is identified to be not irritating.

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:

2017-03-01

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 July 26, 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Abattoir A. Moksel AG, Buchloe, Germany
- Storage, temperature and transport conditions of ocular tissue: Eyes were collected on test day from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
- indication of any existing defects or lesions in ocular tissue samples: The eyes were carefully examined for defects and any defective eyes were discarded.
- Indication of any antibiotics used: Pen/Strep

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20% in physiological saline (0.9% NaCl)

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL
- Lot/batch no. (if required): 609709

Duration of treatment / exposure:

4 h ± 5 min incubation

Duration of post- treatment incubation (in vitro):

90 min

Number of animals or in vitro replicates:

three corneae per dose group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The isolated corneae were obtained from an abattoir. The eyes were collected and subsequently transported in HBSS containing Pen/Strep on ice. Immediately after arrival of the eyes in the laboratory cornea preparation was initiated.

QUALITY CHECK OF THE ISOLATED CORNEAS
The quality of the corneae was checked visually before the tissues were mounted in the corneal holders, they were examined for defects and any defect cornea had been discarded.

NUMBER OF REPLICATES
Three corneae per dose group were used including negative (vehicle) and positive control.

SOLVENT CONTROL USED (if applicable)
yes, vehicle (0.9 % NaCl)

POSITIVE CONTROL USED
yes, 20 % imidazole in 0.9% NaCl

APPLICATION DOSE AND EXPOSURE TIME
750 µl of a solution containing either no substance, 20 % test item or 20 % imidazole were placed in the anterior chamber of the cornea holder for 4 h ± 5 min. After the exposure time the corneas were rinsed at least three times to remove all of the test substance.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes. If YES please specify duration: 90 min

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At least three washing steps wit MEM medium containing phenol red and a final washing step with RPMI medium to substitute the phenol red containing medium.
- POST-EXPOSURE INCUBATION: 90 min

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: yes, using an Opacitometer (BASF-OP3.0, Duratec GmbH)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490)
- Others: pertinent visual observations

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used. - No

Irritation parameter:

in vitro irritation score

Run / experiment:

mean IVIS of three experiments

Value:

9.11

Vehicle controls validity:

valid

Remarks:

IVIS = 0.97 mean value of three corneae

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

IVIS = 153.69 mean value of three corneae

Other effects / acceptance of results:

None of the corneae treated with D-Glucose reaction products with alcohols C16-C18 showed opacity of the tissue.

Results of the Bovine Corneal Opacity and Permeability Assay with 20 % D-Glucose reaction products with alcohols C16-C18 after 4 h exposure

Test group	Cornea No.	Change of Opacity (final - initial opacity)	Corrected Opacity	Permeability at 490 nm	Corrected Permeability at 490 nm	In vitro Irritation Score (mean)	In vitro Irritation Scale
Negative control	1	0.45		0.007			Not severely irritating
	2	0.38		0.009
	3	1 .58		0.018
	MV	0.80		0.011		0.97
Positive control	4	103.36	102.56	3.060	3.049		Severely irritating
	5	99.33	98.52	4.660
	6	81.98	81.18	4.235	4.224
	MV	94.89	94.09	3.985	3.974	153.69
Test Item	7	2.38	1.57	0.332	0.321		Not conclusive
	8	1.69	0.88	0.493	0.482
	9	2.83	2.02	0.733	0.722
	MV	2.30	1.49	0.519	0.508	9.11

Interpretation of results:

study cannot be used for classification

Conclusions:

In the present study the treatment of bovine corneae with D-Glucose reaction products with alcohols C16-C18 for 4h at 32°C resulted in a mean IVIS of 9.11. There were no signs of opacity in none of the treated corneae after 4 h exposure. However, in accordance with OECD Testguideline 437 (adopted July 26, 2013) the following cut-off values for classification according to GHS criteria the IVIS must be < 3 for no classification and > 55 to identify a substance as inducing serious eye damage (UN GHS Category 1). All values between the afore mentioned values do not provide conclusive results, thus, no prediction can be made.

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of D-Glucose reaction products with alcohols C16-C18(20% in 0.9 % NaCl) by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted 26 July 2013.

The corneae were incubated with the test substance and controls for 4 h ± 5 min. After rinsing with MEM and RPMI medium, the corneae were incubated for another 90 min at 32±1°C in order to measure the permeability. The test was performed in triplicates. Opacity and permeability were determined. The in vitro irritancy score (IVIS) were calculated as mean opacity value + (15 x mean OD490 value); a substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.

A 20% dilution of the test substance in physiological saline caused no visually detectable increase of the corneal opacity. However, the calculated mean in vitro irritation score was 9.11.

The positive control (20% imidazole) increased the opacity and permeability of the corneae in both experiments (mean in vitro irritation score 153.69). 

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed in both experiments (mean in vitro irritation score 0.97). Since the mean in vitro irritancy score of the test substance was <55.1 but > 3, no valid conclusion can be drawn after a 4h treatment of bovine corneae with a 20% dilution of D-Glucose reaction products with alcohols C16-C18 in pyhsiological saline under the experimental conditions described in this report.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-04-26 to 2017-04-28

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:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Method Summary No. 164: EpiOcular™ Eye Irritation Test

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) For the prediction of acute ocular irritation of chemicals For use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

other: human tissue model, therefore not applicable

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
This in vitro method is recommended to identify chemicals that do not require classification for eye irritation or serious eye damage according to UN GHS (UN GHS “No Category”) without further testing within a tiered testing strategy from those requiring classification and labelling (UN GHS categories 1 and 2). It therefore can be used for regulatory purposes as an initial step in the Bottom-Up approach or as one of the last steps in a Top-Down approach. It is not intended to differentiate between UN GHS “Category 1” (serious eye damage) and UN GHS “Category 2” (eye irritation) which would require additional testing.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.
Analysis for potential biological contaminants did not reveal any contamination by viruses, bacteria, yeast or other fungi. The certificate of analysis dated 2017-04-25 for Lot Number 23777 (keratinocyte strain 4F1188) is attached to the study report.

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): 50 mg (83.3 mg/cm²)

Duration of treatment / exposure:

6 ± 0.25 h

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

18 ± 0.25 h

Number of animals or in vitro replicates:

2 tissues per dose group

Details on study design:

- Details of the test procedure used: see section “any other information on materials and methods incl. tables”.
- RhCE tissue construct used, including batch number: EpiOcular™ tissue kits (OCL-200-EIT; MatTek) containing 24 inserts with EpiOcular™ tissues on agarose (Lot No.: 23777)
- Doses of test chemical and control substances used: test item 50 mg (83.3 mg/cm²); negative control: 50 μL Aqua dest.; positive control: 50 μL neat methyl acetate.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): exposure: 6 ± 0.25 h at 37 ± 1 °C, 5.0% CO2 / 95% air. Post-exposure immersion: 25 ± 2 min at room temperature. Post-exposure incubation: 18 ± 0.25 h at 37 ± 1°C, 5.0% CO2 / 95% air.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): Positive control: 2. Negative control: 2. NSMTT: two killed tissues were treated with 50 mg of the test item and one tissue was treated with 50 μl of the negative control.
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm using a filter band pass of maximum ± 30 nm.
- Description of the method used to quantify MTT formazan: Per each tissue 2 x 200 μL aliquots of the extract were transferred into a 96-well plate and OD was measured in a plate spectrophotometer using isopropanol as a blank.
- Description of evaluation criteria: The test item is considered to be irritant to the eye if the relative tissue viability is less or equal to 60%. The test item is considered to be non-irritant if relative tissue viability is higher than 60%.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: see “any other information on results incl. tables”.
- Complete supporting information for the specific RhCE tissue construct used: The certificate of analysis dated 2017-04-25 for Lot Number 23777 (keratinocyte strain 4F1188) is attached to the study report and states that the lot was free of contamination and that tissue viability and barrier function were within the acceptable ranges and indicated appropriate formation of the mucosal barrier and a viable basal cell layer.
- Positive and negative control means and acceptance ranges based on historical data: see section “any other information on results incl. tables”.

Test Acceptance Criteria
- 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: tissue viability

Run / experiment:

main experiment

Value:

43.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- Acceptance criteria met for negative control: yes, mean absolute OD570 nm of the negative control is > 0.8 and < 2.5 (measured: 1.180).
- Acceptance criteria met for positive control: yes, mean relative tissue viability of the positive control is < 50% (measured: 5.8).
Max. Difference of % Viability [%] is < 20% (measured 5.3)

Pre-Experiments:

The mixture of 50 mg test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.

The mixture of 50 mg test item per 2 mL isopropanol showed no relevant colouring as compared to the solvent (OD = 0.016). The mixture of 50 mg test item per 1 mL A. dest. showed colouring above 0.08 OD (0.164). Therefore, coloured tissue controls were performed for quantitative correction of results in parallel to the main experiment.

 

Main experiment:

Table 1: Result of the Test Item

Name	Negative Control		Positive Control		Test Item
Tissue	1	2	1	2	1	2
Absolute OD570	1.140	1.183	0.099	0.121	0.578	0.494
	1.160	1.238	0.102	0.116	0.555	0.521
OD570(Blank-Corrected)	1.097	1.140	0.055	0.077	0.534	0.450
	1.116	1.194	0.059	0.073	0.512	0.478
Mean OD570 of the Duplicates (Blank-Corrected)	1.106	1.167	0.057	0.075	0.523	0.464
Total Mean OD570 of 2 Replicate Tissues (Blank-Corrected)	1.137*		0.066		0.493
SD OD570	0.042		0.011		0.037
Relative Tissue Viability [%]	97.3	102.7	5.0	6.6	46.0	40.8
Relative Tissue Viability Difference [%]***	5.3		1.6		5.2
CV [% Viability]	5.3		27.3		12.0
Mean Relative Tissue Viability [%]	100.0		5.8**		43.4
NSCCV	100.0		5.8		40.9

* Corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability

** mean relative tissue viability of the positive control is < 50%

*** relative tissue viability difference of replicate tissues is < 20%.

 

 

Table 2: Test Acceptance Criteria

	Value	Cut off	pass/fail
Mean Absolute OD570 nmNK	1.180	0.8 < NK < 2.5	pass
Mean Relative Viability PC [%]	5.8	< 50%	pass
Max. Difference of % Viability [%]	5.3	< 20%	pass

 

 

Table 3: Historical Data

	Absolute OD570 ± 30 nmNK	Relative Viability PC [%]	Difference of Viability [%]
Mean	1.664	28.9	9.9
SD	0.311	12.0	15.9
n	14	14	53

Historical control data were generated from 2012 – 2016.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

In this study under the given conditions the test item showed irritant effects.

Executive summary:

In this study conducted according to OECD test guideline 492 (adopted July 28, 2015), the potential of the test item to induce eye irritation was analysed by using the three-dimensional human corneal epithelium model EpiOcular, consisting of normal, human-derived epidermal keratinocytes mimicking characteristics of the corneal epithelium.

The test item was applied topically to the EpiOcular tissue for 6 h followed by 25 min post-soaking incubation after removal of the test item. After an 18 h post-treatment period cytotoxic effects were determined via MTT reduction assay.

Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with Aqua dest.

The test item showed no non-specific reduction of MTT and no relevant colouring after mixture with isopropanol but an OD above 0.08 after mixture with Aqua dest. (0.164). Therefore, NSC living was determined to 2.5% in parallel to the main experiment.

The test item showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 60% (43.4%). Therefore, no correction for NSCCV was necessary.

The controls confirmed the validity of the study. The mean absolute OD570 of the two negative control tissues was > 0.8 and < 2.5 (1.180). The mean relative tissue viability

(% negative control) of the positive control was < 50% (5.8%). The maximum inter tissue difference of replicate tissues of all dose groups was < 20% (5.3%).

In this study under the given conditions the test item showed irritant effects.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

In a dermal irritation study performed in accordance with OECD Guideline 439 (In Vitro Skin Irritation) (adopted July 28, 2015), D-Glucose reaction products with alcohols C16-C18 was applied to the three-dimensional human epidermis model tissue for an exposure period of 15 minutes in triplicates. 5 μL of deionised water were topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. Each approximately 10 mg of the test item were applied to the wetted tissues. The test item was spread to match the surface of the tissue.

After 15 minutes exposure at room temperature, the tissues were washed with DPBS to remove any residual test material. Subsequently the tissue constructs were incubated for 42 h at 37 ± 1°C. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive (5% SDS) and negative (aqua dest.) control gave responses that were within the acceptance criteria and as such demonstrated the validity of the study.

The relative mean tissue viability obtained after 15 minutes treatment and 42 h post-incubation with D-Glucose reaction products with alcohols C16-C18 compared to the negative control tissues was 102 %. Since the mean relative tissue viability for the test substance was above 50%, the test item is identified to be not irritating.

 

 

Eye irritation

 An in vitro study was performed to assess the corneal irritation and damage potential of D-Glucose reaction products with alcohols C16-C18 (20% in 0.9 % NaCl) by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted 26 July 2013.

The corneae were incubated with the test substance and controls for 4 h ± 5 min. After rinsing with MEM and RPMI medium, the corneae were incubated for another 90 min at 32±1°C in order to measure the permeability. The test was performed in triplicates. Opacity and permeability were determined. The in vitro irritancy score (IVIS) were calculated as mean opacity value + (15 x mean OD490 value); a substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.

A 20% dilution of the test substance in physiological saline caused no visually detectable increase of the corneal opacity. However, the calculated mean in vitro irritation score was 9.11.

The positive control (20% imidazole) increased the opacity and permeability of the corneae in both experiments (mean in vitro irritation score 153.69). 

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed in both experiments (mean in vitro irritation score 0.97). Since the mean in vitro irritancy score of the test substance was < 55.1 but > 3, no valid conclusion could be drawn after a 4h treatment of bovine corneae with a 20% dilution of D-Glucose reaction products with alcohols C16-C18 in pyhsiological saline under the experimental conditions described in this report.

 

Thus a second in-vitro study according to a top-down approach was performed. In this study conducted according to OECD test guideline 492 (adopted July 28, 2015), the potential of the test item to induce eye irritation was analysed by using the three-dimensional human corneal epithelium model EpiOcular, consisting of normal, human-derived epidermal keratinocytes mimicking characteristics of the corneal epithelium.

The test item was applied topically to the EpiOcular tissue for 6 h followed by 25 min post-soaking incubation after removal of the test item. After an 18 h post-treatment period cytotoxic effects were determined via MTT reduction assay.

 

Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with Aqua dest. The test item showed no non-specific reduction of MTT and no relevant colouring after mixture with isopropanol but an OD above 0.08 after mixture with Aqua dest. (0.164). Therefore, NSC living was determined to 2.5% in parallel to the main experiment. The test item showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 60% (43.4%). Therefore, no correction for NSCCV was necessary. The controls confirmed the validity of the study. The mean absolute OD570 of the two negative control tissues was > 0.8 and < 2.5 (1.180). The mean relative tissue viability (% negative control) of the positive control was < 50% (5.8%). The maximum inter tissue difference of replicate tissues of all dose groups was < 20% (5.3%).

 

In this study under the given conditions the test item showed irritant effects.

 

Respiratory irritation

No data on the respiratory irritation of D-Glucose, reaction products with alcohols C16-18 (even numbered) are available.

 

There are no data gaps for the endpoint irritation/corrosion. No human information is available for this endpoint. However, there is no reason to believe that these results would not be applicable to humans.

Justification for classification or non-classification

Skin irritation

Based on reliable, adequate and relevant data, D-Glucose, reaction products with alcohols C16-18 (even numbered) does not need to be classified for skin irritation according to regulation (EC) 1272/2008.

 

Eye irritation

Based on reliable, adequate and relevant data, D-Glucose, reaction products with alcohols C16-18 (even numbered) has to be classified as Category 2, irritating to eyes according to CLP, EU GHS (Regulation (EC) No 1272/2008) and is assigned the hazard statement H319 and the signal word “Warning”.