1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl 4-chloro-4-deoxy-α-D-galactose

Endpoint summary

Description of key information

To determine whether the test item causes serious skin damage or requires classification for skin irritation, a GLP study according to OECD 439 was carried out. Under the conditions of the test assay, the test article was not considered to be irritant in the in vitro skin model EpiDerm SIT (EPI-200).

To determine whether the test item causes serious eye damage or requires classification for eye irritation, a GLP study according to OECD 437 was carried out. Under the conditions of this assay, the test substance produced an IVIS score of -0.04 and does not require classification for eye irritation.

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:

10 January 2018 - 01 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: MC16B92011
- Expiration date of the lot/batch: 31 Jan 2018
- Purity test date: 01 Feb 2016; 100.1%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15-25 degrees Celsius, protected from light

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: reconstructed epidermis with a functional stratum corneum, supplied by MatTek In Vitro Life Science Laboratories, Bratislava, Slovak Republic

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm SIT (EPI-200)
- Date of initiation of testing: 10 January 2018
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 +/- 1 degrees Celsius
- Temperature of post-treatment incubation (if applicable): 37 +/- 1 degrees Celsius
REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After 1 hour treatment, tissues were washed with PBS and then placed on appropriate medium and incubated for 42 hours. After 42 hour recovery period, each tissue was rinsed again with PBS (no volumes specified).
- 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: 0.3 mL of 1 mg/mL MTT in PBS
- Incubation time: 3 hours
- Spectrophotometer: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Quantified using MTT; Viability of tissues treated with positive control should be less than or equal to 20%.
- Barrier function: Stratum corneium is robust to resist the rapid penetration of 5% SDS.
- Contamination: None
- Reproducibility: Yes

NUMBER OF REPLICATE TISSUES:
3
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE : Not applicable

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
1
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
-Test substances are considered to be irritant to skin in accordance with UN GHS Category 2 if the tissue viability after exposure and post-treatment incubation is less than or equal to 50%, relative to negative control
-Test substances are considered to be non-irritant to skin in accordance with UN GHS if the tissue viability after exposrue and post-treatment incubation is greater than 50%, relative to negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

VEHICLE
- Amount(s) applied (volume or weight with unit): No vehicle used with test substance; Tissue was moistened with 25 ul sterile PBS before application of test substance.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 ul

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 ul

Duration of treatment / exposure:

Treated tissues were placed into an incubator for 35 minutes. The plates were removed from the incubator and placed into a sterile hood until the 60 minute treatment period was complete for each tissue. Following treatment, substances were removed by washing the tissues with PBS.

Duration of post-treatment incubation (if applicable):

Following treatment, the washed tissues were placed on appropriate medium and incubated for 42 hours.

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Replicate A

Value:

91.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Replicate B

Value:

95.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Replicate C

Value:

113.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

100.1

Vehicle controls validity:

not examined

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: None
- Direct-MTT reduction: None
- Colour interference with MTT: None

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes; the OD values for negative controls were greater or equal to 0.8 and less than or equal to 2.8.
- Acceptance criteria met for positive control: Yes; Group mean viability for positive control was 3.7%.
- Acceptance criteria met for variability between replicate measurements: Yes

The group mean viability for the test article was 100.1%. The group mean viability for the positive control was 3.7%.

Interpretation of results:

GHS criteria not met

Conclusions:

To determine whether the test item causes serious skin damage or does not require classifying for skin irritation a GLP study according to OECD 439 was carried out. Under the conditions of the test assay, the test article was not considered to be irritant in the in vitro skin model EpiDerm SIT (EPI-200).

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:

19 December 2017 - 01 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

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)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: MC16B92011
- Expiration date of the lot/batch: 31 Jan 2018
- Purity test date: 01 Feb 2018; 100.1%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15-25 degrees Celsius, protected from light

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: 20% w/v solution in 0.9% sodium chloride solution

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Local abattoir
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were removed after slaughter, completely immersed in Hank's Balanced Salt Solution (containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL) and transported on same day to testing facility. Upon arrival at test facility, corneas were excised from the eyes and loaded onto holders. Both chambers of each holder were filled with Minimal Essential Medium (MEM) and incubated at 32+/- 1 degrees Celsius for at least 1 hour.
- Time interval prior to initiating testing: Within 24 hours after receipt at test facility
- indication of any existing defects or lesions in ocular tissue samples: On arrival at the test facility the eyes were carefully examined for defects including increased opacity, scratches and neovascularisation. Only corneas free from such defects were used.; opacity was measured using opacitometer to look for scratches or increased neovascularization
- Indication of any antibiotics used: Yes; 100 IU/mL penicillin and 100 ug/mL streptomycin

Vehicle:

other: 0.9% sodium chloride solution

Remarks:

Test substance was dissolved in sodium chloride but study indicated that this was not a vehicle control but a negative control.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

1 hour 25 minutes

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Corneas were excised from the eyes and loaded onto specifically designed holders. Both chambers of each holder (posterior and anterior) were filled with warmed Minimal Essential Medium (MEM). Holders were incubated at 32 +/- 1 degress Celsius for at least 1 hour. After incubation, the media was removed from both chambers and fresh media was added.
QUALITY CHECK OF THE ISOLATED CORNEAS
The opacity of each cornea was measured using an opacitometer. Any corneas found to have scratches, increased neovascularization or an opacity of > 7 opacity units when examined prior to treatment were discarded.
NUMBER OF REPLICATES
3
NEGATIVE CONTROL USED
0.9% sodium chloride solution
SOLVENT CONTROL USED (if applicable)
None
POSITIVE CONTROL USED
20% w/v Imidazole in 0.9% sodium chloride solution
APPLICATION DOSE AND EXPOSURE TIME
750 µL of test material was applied to each of 3 corneas followed by 4 hour incubation at 32 degrees Celsius
TREATMENT METHOD: open chamber
POST-INCUBATION PERIOD: Yes. If YES please specify duration
1 hour and 25 minutes
REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Each cornea was washed with media containing phenol red (as a pH indicator) until the indicator showed no pH effect occurring. The corneas were then washed once in media without phenol red so that corneal opacity could be measured.
- POST-EXPOSURE INCUBATION:
After washing to remove test substance, each cornea was then incubated in vertical position in media and sodium fluroescein for 1 hour and 25 minutes.
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer
- Corneal permeability: Passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490)
- Others (e.g, pertinent visual observations, histopathology): Visual, but no histopathology was conducted

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IIVS = mean opacity value + (15 X mean permeabiliity value)
DECISION CRITERIA:
-Serious eye damage if IVIS greater than equal to 55
-Not classified for eye irritation if IVIS is equal to or less than 3
-If IVIS is greater than 3 and less than or equal to 55, no prediction can be made.

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean

Value:

-0.003

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

0

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

-0.04

Vehicle controls validity:

not examined

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:
Yes
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline: OK

Interpretation of results:

GHS criteria not met

Conclusions:

To determine whether the test item causes serious eye damage or does not require classifying for eye irritation a GLP study according to OECD 437 was carried out. Under the conditions of this assay, the test substance produced an IVIS score of -0.04 and does not require classification for eye irritation.

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

According to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments), the test substance should be classified as non-irritating to eyes and skin.