Registration Dossier

Administrative data

Description of key information

- skin irritation: guideline study, OECD 439, GLP, in vitro: not irritating

- eye irritation: guideline study, OECD 437, GLP, in vitro: not 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:
2017-03-06 to 2017-05-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
July 28, 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
July 06, 2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
This test method provides an in vitro procedure that, depending on information requirements, may allow determining the cytotoxic potency and skin irritancy of test items as a stand-alone replacement test within a testing strategy, in a weight of evidence approach. The procedure described under this test method allows the hazard identification of irritant substances in accordance with UN GHS Category 1 or Category 2.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epiderm™
- Tissue batch number(s): EPI-200, Lot no. 25803
- Date of initiation of testing: 2017-03-14

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C for 35 min and room temperature for the last 25 min


REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: not reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3h
- Spectrophotometer: Tecan Sunrise Magellan Version 7.2
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The preferred assay for determining the magnitude of viability is the MTT assay. The optical density (OD) of the extraction solvent alone should be sufficiently small, i.e. OD < 0.1. The tissue treated with the negative control (NC) should exhibit stability in culture (provide similar viability measurements) for the duration of the test exposure period.

- Barrier function: The EpiDerm™ System was manufactured according to defined quality assurance procedures. All biological components of the epidermis and the culture medium were tested by manufacturer for viral, bacterial, fungal and mycoplasma contamination. MatTek Corporation determines the ET 50 value following exposure to Triton X-100 (1%) for each EpiDerm lot. The ET 50 must fall within a range established based on a historical database of results. ET-50 assay: 100µL 1% Triton x-100, 4 time points, n = 3, MTT assay: Acceptance criteria: ET-50 [4.77-8.72], Result: 5.36h.
- Contamination: HIV-1, Hepatitis B virus, Hepatitis C virus, Bacteria, yeast and other fungi - longterm antibiotic, antimycotic free culture: Not detected


NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues
- N. of replicates : 3

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-irritant to skin if the viability after exposure 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): 25 mg to cover the skin uniformly with an area of 0.63 cm². The test item is a fine powder. For better contact of the test item to the skin, the skin surface was moistened with 25 µL Dulbecco’s phosphate buffered saline.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): Dulbecco's phosphate buffered saline (D-PBS) 30µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): aqueous sodium dodecyl sulphate (SDS) 30 µL
- Concentration (if solution): 5%
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
42h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
test substance
Value:
117.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
optical density was 1.418
Positive controls validity:
valid
Remarks:
viability 6.1% of the negative control
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: Not reported
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute OD of the negative control (NC) tissues (treated with sterile PBS buffer) in the MTT test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use. The assay meets the acceptance criterion if the mean OD of the NC tissues is ≥ 1.0 and ≤ 2.5. The mean optical density (OD) of 3 negative control tissues was 1.418.

- Acceptance criteria met for positive control: The assay meets the acceptance criterion if the mean viability of PC tissues expressed as % of the negative control tissues is ≤ 20%. The viability of cells treated with the positive reference item, 5% SDS, was 6.1% of the negative control and fulfilled the acceptance criterion of ≤ 20%.
- Acceptance criteria met for variability between replicate measurements: The assay meets the acceptance criterion if the standard deviation (SD) calculated from individual % tissue viabilities of the 3 identically treated replicates is ≤ 18%. The standard deviation determined for all triplicates was below the limit of acceptance of 18%.

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour. Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

Table 1: Summary of results

Group

Absorbance 570 nm Tissue 1

Absorbance 570 nm Tissue 2

Absorbance 570 nm Tissue 3

Mean

Absorbance

of 3 Tissues

Relative

Standard

Deviation

[%]

Rel. Absorbance

[% of

Negative

Control]

Negative control

1.503

1.319

1.431

1.418

6.6

100.0

Positive control

0.092

0.083

0.085

0.087

5.3

6.1

Test item

1.557

1.624

1.807

1.663

7.8

117.2

Interpretation of results:
GHS criteria not met
Conclusions:
In a study with alpha methyl glucoside conducted according to OECD guideline 439, the skin irritation was determined in vitro using the reconstructed human epidermis model EPIDERM ™. The cell viability was determined using the MTT assay. Since the cell viability was 117.2 % in this test and all validity criteria were fulfiled alpha methyl glucoside is considered to be non-irritating to the skin.
Executive summary:

In a study with alpha methyl glucoside conducted according to OECD guideline 439, the skin irritation was determined in vitro using the reconstructed human epidermis model EPIDERM ™. The three-dimensional human epidermis model tissue was exposed to 25 mg of the test substance on a surface area of 0.63cm² in triplicates for 60 min and subsequently washed and incubated for further 42h. 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 (deionised water) 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 60 minutes treatment with alpha methyl glucoside compared to the negative control tissues was 117.2 %. Since the mean relative tissue viability for the test substance was above 50%, alpha methyl glucoside is identified to be not irritating.

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:
2017-03-06 to 2017-05-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
July 26, 2013
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
December 09, 2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Hubert Bahlmann GmbH & Co. Versandschlachterei Spezialmischfutterwerk KG, 49699 Lindern, Germany
- Number of animals: at least five, three corneae for each concentration (negative control, positive control, treatment)
- Characteristics of donor animals (e.g. age, sex, weight): between 6 and 12 month old cattle
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): To minimize deterioration and bacterial contamination, on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution 2 (HBSS) containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL.
- indication of any existing defects or lesions in ocular tissue samples: Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation. Only corneas from eyes free of defects were used.
- Indication of any antibiotics used: penicillin at 100 IU/mL and streptomycin at 100 µg/mL
Vehicle:
unchanged (no vehicle)
Remarks:
20% suspension in 0.9% sodium chloride solution (w/v)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20% suspension in 0.9% sodium chloride solution (w/v)

Duration of treatment / exposure:
240 min
Duration of post- treatment incubation (in vitro):
90 ± 5 min
Number of animals or in vitro replicates:
three corneae per treatment
Details on study design:
SELECTION AND PREPARATION OF CORNEAE
Bovine eyes from cattle in the age range of 6 to 12 months were obtained from a slaughterhouse. To minimize deterioration and bacterial contamination, on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution 2 (HBSS) containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL 3 . Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation. Only corneas from eyes free of defects were used.

NUMBER OF REPLICATES
Three coneae per concentration

SOLVENT CONTROL USED (if applicable)
0.9 % sodium chloride solution (w/v)

POSITIVE CONTROL USED
20% Imidazole in 0.9% sodium chloride solution

APPLICATION DOSE AND EXPOSURE TIME
750 µL of each solution was applied for 240 min

TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least three times

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The IVIS cut-off values for identifying test chemicals as inducing serious damage (UN GHS Category 1) and test chemicals not requiring classification for irritation or serious eye damage (UN GHS No Category):
≤ 3: No Category
> 3 and ≤ 55: No prediction can be made
> 55: Category 1
Irritation parameter:
in vitro irritation score
Run / experiment:
1
Value:
2.097
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
1
Value:
-1.99
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein retention score
Run / experiment:
1
Value:
-0.007
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The negative or solvent/vehicle control responses should result in opacity and permeability values, that are less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control. (see Table 1)
- Acceptance criteria met for positive control: Yes. The positive control should give an IVIS that falls within two standard deviations of the current historical mean. (see Table 1)
- Range of historical values if different from the ones specified in the test guideline: No

Table 1: Historical control data

 

Parameter

Mean

Standard deviation

Lower limit of acceptance

(mean-2*SD)

Upper limit of acceptance

(mean+2*SD)

NaCl 0.9 %

IVIS Opacity

0.812 0.513

0.762 0.788

-0.712 -1.064

2.336 2.089

 

Permeability

0.020

0.012

-0.005

0.045

Imidazol

20%

IVIS Opacity

86.039

63.051

10.607

10.709

64.826

41.634

107.253

84.468

 

Permeability

1.533

0.463

0.607

2.458

Table 2: Summary of results after 240 min incubation

Test group

Opacity

Permeability at 490 nm

In vitro Irritation score

Mean In vitro Irritation Score

In vitro Irritation Scale

Negative control

0.917

0.010

1.067

 

Not severely irritating

-0.797

0.011

-0.632

 

-0.358

0.010

-0.208

0.076

Positive control

73.505

1.403

94.629

 

Severely irritating

49.323

1.204

67.462

 

54.463

1.372

75.122

79.071

Test item

-2.032

0.006

-2.043

 

Not severely irritating

-2.031

0.005

-2.027

 

-2.151

-0.010

-2.222

-2.097

Interpretation of results:
GHS criteria not met
Conclusions:
In the present study with alpha methyl glucoside conducted according to OECD guideline 437, three corneae for each treatment, i.e. negative control, positive control (20% imidazol in 0.9 % sodium chloride solution (w/v)) and treatement (20% alpha methyl glucoside in 0.9 % sodium chloride solution (w/v)) were exposed for 240 min. Opacity and permeability revealed values < 0 and the IVIS was < 3 (2.097), therefore, the test item is not classified according to UN GHS classification.
Executive summary:

In the present study with alpha methyl glucoside conducted according to OECD guideline 437, three corneae for each concentration, i.e. negative control, positive control (20% imidazol in 0.9 % sodium chloride solution (w/v)) and treatement (20% alpha methyl glucoside in 0.9 % sodium chloride solution (w/v)) were exposed for 240 min. Subsequently the opacity was determined using a opacitometer and the corneal permeability was detected with the fluorescein method.

The positive control increased opacity and permeability (mean in vitro score 79.07) corresponding to a classification as irreversible effects to the eye.

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean in vitro score 0.00).

Since, the results of the test item for opacity and permeability revealed values < 0 and the IVIS was < 3 (2.097) the test item is not classified according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

- skin irritation:

In a study conducted according to OECD guideline 439, the skin irritation was determined in vitro using the reconstructed human epidermis model EPIDERM ™. The three-dimensional human epidermis model tissuewas exposed to 25 mg of the test substance on a surface area of 0.63cm² in triplicates for 60 min and subsequently washed and incubated for further 42h.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 (deionised water) 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 60 minutes treatment with alpha methyl glucoside compared to the negative control tissues was 117.2 %. Since the mean relative tissue viability for the test substance was above 50%, alpha methyl glucoside is identified to be not irritating.

- eye irritation:

In the present study conducted according to OECD guideline 437, three corneae for each treatment, i.e. negative control, positive control (20% imidazol in 0.9 % sodium chloride solution (w/v)) and treatement (20% alpha methyl glucoside in 0.9 % sodium chloride solution (w/v)) were exposed for 240 min. Subsequently, the opacity was determined using a opacitometer and the corneal permeability was detected with the fluorescein method.

The positive control increased opacity and permeability (mean in vitro score 79.07) corresponding to a classification as corrosive to the eye.

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean in vitro score 0.00).

Since, the results of the test item for opacity and permeability revealed values < 0 and the IVIS was < 3 the test item is considered to be not severe irritant or not corrosive.

Justification for classification or non-classification

Based on reliable, adequate and relevant data the substance does not need to be classified according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS) with repsect to skin/eye irritation or corrosion.