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

Skin irritation / corrosion

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2009

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

259-952-2

EC Name:

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

Cas Number:

56038-13-2

Molecular formula:

C12H19Cl3O8

IUPAC Name:

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

Test material form:

solid: crystalline

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

In vitro test system

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

Results and discussion

In vitro

Results

Results 1

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

Results 2

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

Results 3

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

Results 4

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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).