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Description of key information

standard in vitro tests according to OECD ITS available.

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:
09.10.17-13.10.17
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:
2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: CF1001
- Expiration date of the lot/batch:
- Purity test date:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity:
- Specific activity:
- Locations of the label:
- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
commercially available EpiDermTM-Kit.
The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cul-tured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers representing main
lipid classes analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell culture inserts.

Origin
EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava.
Designation of the kit: EPI-200-SIT
Day of delivery: 10. Oct. 2017
Batch no.: 25848
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
Amount Test item
#01: 25.0 mg
#02: 25.7 mg
#03: 24.9 mg

Negative control: 30 µL DPBS buffer per tissue
Positive control: 30 µL 5 % SDS solution per tissue
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
23 h 25 min
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
#01
Value:
90.2
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:
#02
Value:
96.2
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:
#03
Value:
94.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The mean value of relative tissue viability of the test item was reduced to 93.8% after the treat-ment. This value is above the threshold for skin irritation (50%). Therefore, the test item is considered as non-irritant to skin.
Interpretation of results:
GHS criteria not met
Conclusions:
The test item is considered to be not skin irritant.
Executive summary:

One valid experiment was performed.

Three tissues of the human skin model EpiDermTMwere treated with2,2'-Azobis(2,4-dimethylvaleronitrile)for 60 minutes.

The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2; as indicated by the supplier).

DPBS-buffer was used as negative control and 5 % SDS solution was used as positive control.

After treatment with the negative control, the mean absorbance value was within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.9. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 1.7% (required:£20 %).

The variation within the tissue replicates of negative, control, positive control and test item was acceptable (required: ≤ 18 %).

 

After the treatment with the test item, the mean value of relative tissue viability was reduced to 93.8 %. This value is above the threshold for skin irritation potential (50 %). Test items that induce values above the threshold of 50 % are considered non-irritant to skin.

 

Therefore,2,2'-Azobis(2,4-dimethylvaleronitrile)is considered

non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23.11.17
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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
cattle
Strain:
other: Bos primigenius Taurus
Details on test animals or tissues and environmental conditions:
Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Peni-cillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container.
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): #01: 383.4 mg; #02: 386.5 mg; #03: 393.8 mg
- Concentration (if solution):-

VEHICLE
- Amount(s) applied (volume or weight with unit): -
- Concentration (if solution):-
- Lot/batch no. (if required): -
- Purity:-
Duration of treatment / exposure:
Exposure time on the corneas was 4 hours at 32 ± 1 °C.
Number of animals or in vitro replicates:
three replicates per treatment group
Irritation parameter:
in vitro irritation score
Run / experiment:
#01
Value:
0.54
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
#02
Value:
0.34
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
#03
Value:
-0.34
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Values for negative and positive controls were within the range of historical data of the test facility. Therefore, the test system was acceptable.
Interpretation of results:
GHS criteria not met
Conclusions:
The test item 2,2'-Azobis(2,4-dimethylvaleronitrile) showed no effects on the cornea of the bo-vine eye. The calculated IVIS (In Vitro Irritancy Score) is 0.18.
Executive summary:

One valid experiment was performed.

Bovine corneas were used. They were collected from slaughtered cattle, which were between 12 and 60 months old.

The test item2,2'-Azobis(2,4-dimethylvaleronitrile)was brought onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured.

The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured.

HBSS was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (In VitroIrritancy Score) is 2.51.

20 % imidazole solution was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated IVIS (In VitroIrritancy Score) is 120.59.

 

Under the conditions of this study, the test item2,2'-Azobis(2,4-dimethylvaleronitrile)showed no effects on the cornea of the bovine eye. The calculated IVIS (In VitroIrritancy Score) is 0.18.

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15. January - 02. May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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)
GLP compliance:
yes (incl. certificate)
Species:
human
Details on test animals or tissues and environmental conditions:
Specification
Commercially available EpiOcularTM kit.
The EpiOcularTM tissue consists of normal, human-derived keratinocytes which have been
cultured to form a stratified squamous epithelium similar to that found in the human cornea.
It consists of highly organized basal cells. These cells are not transformed or transfected
with genes to induce an extended life span. The EpiOcularTM tissues are cultured in
specially prepared cell culture inserts with a porous membrane through which nutrients
can pass to the cells. The tissue surface is 0.6 cm2.

Origin
EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories,
Mylnské Nivy 73, 82105 Bratislava, Slovakia.
Designation of the kit: OCL-200-EIT
Day of delivery: 16. Jan. 2018
Batch no.: 27019
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
50 mg
Duration of treatment / exposure:
6 h
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
2
Details on study design:
Preparations
On the day of the start of the experiment, the MTT concentrate was thawed. The MTT concentrate was diluted with assay medium directly before use. The assay medium was warmed in the water bath to 37 ± 1 °C. 6-well-plates were labelled with test item, negative control and positive control and filled with 1 mL assay medium in the appropriate wells. All inserts were inspected for viability and the presence of air bubbles between agarose gel and insert. Viable tissues were transferred in the prepared 6-well-plate and incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 1 hour. After the pre-incubation, the medium was replaced and the wells were filled with 1 mL fresh assay medium. All 6-well-plates were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 16 hours.

Exposure and Post-Treatment
After overnight incubation, the tissues were pre-wetted with 20 μL DPBS buffer and the tissues were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 30 minutes. After that, 50 μL of the controls and a defined amount of the test item were applied in duplicate in one- minute- intervals. At the beginning of each experiment (application of negative controls), a stop watch was started. After dosing the last tissue, all plates were transferred into the incubator for 6 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At the end of exposure time, the inserts
were removed from the plates in one-minute-intervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately transferred into 5 mL of assay medium in pre-labelled 12-well plate for 25 minutes post soak at room temperature.
After that, each insert was blotted on absorbent material and transferred into a pre-labelled 6-well plate, containing 1 mL assay medium. For post-treatment incubation, the tissues were incubated for 18 hours 10 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity.
After the post-treatment incubation, the MTT assay was performed.

MTT Assay and Extraction
A 24-well-plate was prepared with 300 μL freshly prepared MTT solution in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solution. The plate was incubated for 180 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity.
At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, containing 2 mL isopropanol, taking care that no isopropanol is flowing into the tissue insert. The plate was firmly sealed to avoid evaporation of the solvent and then shaken for 2 hours at room temperature, protected from light.

Measurement
The inserts were removed from the 6-well plate and discarded. The content of each well was thoroughly mixed in order to achieve homogenisation.
From each well, two replicates with 200 μL solution (each) were pipetted into a 96-wellplate.
Eight wells with 200 μL isopropanol were pipetted also. The plate was read in a plate spectrophotometer at 570 nm
Irritation parameter:
other: % viability
Run / experiment:
mean
Value:
105.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the test, 2,2'-Azobis(2,4-dimethylvaleronitrile) is considered
non-eye irritant in the EpiOcularTM Eye Irritation Test.
Executive summary:

One valid experiment was performed.

The test item 2,2'-Azobis(2,4-dimethylvaleronitrile) was applied to a three-dimensional human

cornea tissue model in duplicate for an exposure time of 6 hours.

The solid test item was applied to two tissue replicates.

After treatment, the respective substance was rinsed from the tissue; then, cell viability of

the tissues was evaluated by addition of MTT, which can be reduced to formazan. The

formazan production was evaluated by measuring the optical density (OD) of the resulting

solution.

Demineralised water was used as negative control and methyl acetate was used as positive

control.

The controls showed the following results: After treatment with the negative control, the

absorbance values were within the required acceptability criterion of mean OD > 0.8 and <

2.5, OD was 1.7. The positive control showed clear eye irritating effects, mean value of the

relative tissue viability was 31.5 % (< 50%).

Variation within tissue replicates was acceptable (< 20%).

After treatment with the test item, the mean value of relative tissue viability was 105.3 %.

This value is above the threshold for eye irritation potential (≤ 60%).

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

Additional information

Justification for classification or non-classification

the available information is conclusive but not sufficient for classification as either skin or eye irritation