D-cellobiose

Endpoint summary

Description of key information

In a GLP-compliant study according to OECD Guideline 439 , the test substance did not reduce cell viability (100% with an SD of 1.2). Therefore, it is considered not irritating to skin.

In a valid experimental study according to OECD test guideline 492 under GLP conditions, the test substance marginally reduced the cell viability (90%). Therefore, it is considered as not irritating to the eye.

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

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:

version of June 14, 2021

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

version of June 9, 2012

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Pfeifer & Langen GmbH & Co. KG; Lot No. L1021174
- Purity, including information on contaminants, isomers, etc.: 99.54 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light
- Expiry date: 28 October 2023

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): none

Test system:

human skin model

Source species:

human

Vehicle:

unchanged (no vehicle)

Details on test system:

TISSUE
- Model used: EPISKIN(TM) Small Model (SM)
- Tissue batch number(s): 21-EKIN-050
- Production date: not specified
- Shipping date: not specified
- Delivery date: not specified
- Date of initiation of testing: Dec 20, 2021

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C for 42 hours

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 mL PBS solution used to thoroughly rinse the tissue once
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of 0.3 mg/mL MTT per well
- Incubation time: 3 hours +/- 5 min
- Spectrophotometer: not specified
- Wavelength: 570 nm
- Filter: not specified
- Filter bandwidth: not specified
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA: secured to batch release quality control

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: no direct MTT interference

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be non-irritating to skin if the viability is greater than or equal to 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 mg (to moistened skin)

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5% aq.

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:

1

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:

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: -

Interpretation of results:

GHS criteria not met

Conclusions:

In a GLP-compliant study according to OECD Guideline 439 , the test substance did not reduce cell viability (100% with an SD of 1.2). Therefore, it is considered not irritating to skin.

Executive summary:

The skin irritation potential of the test substance was investigated in an experimental study according to OECD test guideline 439 under GLP conditions using the EPISKIN^TMSM. The EPISKIN^TMSM is provided in kits, which are manufactures according to defined quality assurance procedures.

The testing laboratory, which had demonstrated its technical proficiency in conducting the test, received the kit used for testing in good order.

The test substance was applied (10 mg) for 15 minutes to three tissues. After thorough rinsing, the treated tissue were incubated for 42h at 37°C. Then the MTT test was performed, followed by formazan extraction and cell viability measurement with a spectrometer at 570 nm.

Concurrent negative and positive control fulfilled all acceptance criteria, as did the test substance, which did not directly reduce MTT.

With a mean cell viability of 100% (as compared to the negative control), the test substance did not reduce tissue viability.

Using the standard prediction (with a threshold of 50%) the test substance is considered to be not skin irritating and does not require to be classified (UN GHS 'No Category').

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:

from Dec 6, 2021, to Feb 14, 2022

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:

version of June 18, 2019

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Pfeifer & Langen GmbH & Co. KG; Lot No. L1021174
- Purity, including information on contaminants, isomers, etc.: 99.54 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light
- Expiry date: 28 October 2023

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): none

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The EpiOcularTM Eye Irritation Test (EIT) was validated and is scientifically valid 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. Solids are within the applicability domain of the test method

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
EpiOcularTM (OCL-200-EIT)
The EpiOcularTM human cell construct (MatTek In Vitro Life Science Laboratories) is used in this assay. This three-dimensional human cornea model allows the identification of test items with the potential to induce eye irritation or serious eye damage by assessing cell viability after treatment. The model is composed of stratified human keratinocytes in a three- dimensional structure, consisting of at least three viable layers of cells. Test materials can be applied topically to the model so that water insoluble materials may also be tested. Prior to use, each plate (6, 12, and 24-well) and its cover was uniquely identified with a permanent marker by a plate number and/or test item number.
The cytotoxicity of the test item (and thus the ocular irritation potential) is evaluated by the relative viability of the treated tissues in comparison to the negative control-treated tissues. Viability is determined by the NAD(P)H-dependent microsomal enzyme reduction of MTT (and to a lesser extent, by the succinate dehydrogenase reduction of MTT) in control and test item-treated cultures (Berridge, et al., 1996). Data are presented in the form of relative survival (relative MTT conversion).
Supplier: MatTek In Vitro Life Science Laboratories Mlynské Nivy 73, Bratislava, Slovakia
Lot No.: 34945
Expiry date: 10 December 2021

-Demonstration of Proficiency
Prior to routine use of the method TOXI-COOP ZRT. Demonstrated the technical proficiency in a separate study (Study No.: 392-492-1722) using the fifteen Proficiency Chemicals according to OECD Test Guideline No. 492.

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): approx. 50 mg/tissue
- Concentration (if solution): not applicable

Duration of treatment / exposure:

6h

Duration of post- treatment incubation (in vitro):

18h

Number of animals or in vitro replicates:

2 tissues

Details on study design:

- Details of the test procedure used: The test was conducted according to the OCED TG 492 (details below)
- RhCE tissue construct used, including batch number: Kit OCL-200; Lot no.: 34945
- Doses of test chemical and control substances used: approx. 50 mg/tissue
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): Exposure of 6h at approx. 37°C; 25 min post soak at room temperature; and 18h post-incubation at approx. 37°C
- Justification for the use of a different negative control than ultrapure H2O (if applicable): not applicable
- Justification for the use of a different positive control than neat methyl acetate (if applicable): not applicable
- Description of any modifications to the test procedure: none
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): In a pre-test, no indication for direct MTT reduction or colouring by the test substance was observed.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): 2
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm
- Description of the method used to quantify MTT formazan: spectrophotometer
- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable): not applicable
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: The evaluation criteria of the OECD TG 492%, i.e. a threshold of 60% to discriminate non eye irritants from eye irritants, were used.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Both the responses of the negative and the positive controls were within the range of the historical data.
- Complete supporting information for the specific RhCE tissue construct used: see above
- Reference to historical data of the RhCE tissue construct: Acceptable OD range of negative control: 1.094 - 2.609; acceptable relative viability range of the positive control: 2 - 41%
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: Prior to routine use of the method TOXI-COOP ZRT. Demonstrated the technical proficiency in a separate study (Study No.: 392-492-1722) using the fifteen Proficiency Chemicals according to OECD Test Guideline No. 492.
- Acceptable variability between tissue replicates for positive and negative controls: Yes (5% and 1%)
- Acceptable variability between tissue replicates for the test chemical: Yes (5%)

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Experiment 1

Value:

90

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Interpretation of results:

GHS criteria not met

Conclusions:

In a valid experimental study according to OECD test guideline 492 under GLP conditions, the test substance marginally reduced the cell viability (90%). Therefore, it is considered as not irritating to the eye.

Executive summary:

The eye irritation potential of the test substance was investigated in an experimental study according to OECD test guideline 492 under GLP conditions using the EpiOcular^TM tissue. The EpiOcular^TM tissue is provided in kits, which are manufactures according to defined quality assurance procedures.

The testing laboratory, which had demonstrated its technical proficiency in conducting the test, received the kit used for testing in good order.

The test substance was applied (approx. 50 mg) for six hours to two tissues. After thorough rinsing, 25 min post soak at room temperature was performed, before the tissues were post-incubated for 18h post-incubation at approx. 37°C. Then the MTT test was performed and cell viability was measured with a spectrometer at 570 nm.

Concurrent negative and positive control fulfilled all acceptance criteria, as did the test substance, which did not directly reduce MTT, nor was colour interfering.

The test substance marginally reduced the cell viability as compared to the negative control (90.0%).

The results obtained from the study indicated that the test item reveals no eye irritation potential under the applied testing conditions. According to the current OECD Guideline No. 492, cellobiose is, thus, considered as non-irritant to eye (UN GHS No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In a GLP-compliant study according to OECD Guideline 439 , the test substance did not reduce cell viability (100% with an SD of 1.2). Therefore, it is considered not irritating to skin.

In a valid experimental study according to OECD test guideline 492 under GLP conditions, the test substance marginally reduced the cell viability (90%). Therefore, it is considered as not eye irritating to the eye and no classification is warranted.