Registration Dossier

Administrative data

Description of key information

skin irritation: not irritating (OECD 439, GLP)

eye irritation: not irritating (equivalent or similar to OECD 405; non-GLP)

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:
2017-08-09 to 2017-08-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015-07-28
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012-07-06
Deviations:
no
Qualifier:
according to
Guideline:
other: MatTek Corporation Protocol for: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT)
Version / remarks:
2014-11-07
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2015-06-05
Test material information:
Composition 1
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature; protected from light; dry in closed containers
Test system:
human skin model
Source species:
human
Cell type:
other: normal, human epidermal keratinocytes
Cell source:
other: humans
Source strain:
other: not applicable
Details on animal used as source of test system:
not applicable
Justification for test system used:
This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and resembles the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.
Vehicle:
other: Dulbecco's phosphate buffered saline
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (MatTek)
- Tissue lot number: 25835

TEST FOR DIRECT MTT REDUCTION
- to check the non-specific MTT-reducing capability of the test item 25 mg of the test item was mixed per 1 mL MTT medium and incubated for 60 min at 37 ± 1 °C in the incubator.
- if the mixture turns blue/purple, the test item is presumed to have reduced MTT and the part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using killed tissues.

TEST FOR COLOUR INTERFERENCE
- to check the colouring potential of the test item 25 mg of the test item was mixed per 300 µL aqua dest. and per 300 µL isopropanol each in a transparent recipient and incubated at 37 ± 1°C for 60 min.
- if the test item is classified as non-irritant and colouring is detected by unaided eye-assessment, and the chemical in water and/or isopropanol absorbs light in the range of 570 ± 30 nm, the test item was checked for its tissue-colouring potential using additional living tissues treated with the test item.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C, 5.0 % CO2 for 35 ± 1 minutes followed by incubation at room temperature until the 60 ± 1 minute treatment period was completed
- Temperature of post-treatment incubation: 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- after the end of the treatment period the tissues were washed 15 times with DPBS.
- subsequently, the inserts were submerged three times in DPBS and shaken to remove rests of the test item.
- then inserts were rinsed once from the inside and the outside with sterile DPBS.
- inserts were placed in prepared 6-well plates containing pre-warmed fresh assay medium per well.
- plates were post-incubated at 37 ± 1 °C, 5.0% CO2, humidified to 95%, for 24 ± 2 h. Following this incubation the tissues were transferred to new wells containing fresh assay medium and incubated for additional 18 ± 2 h.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL (300 µL/well)
- Incubation time: 3 hours ± 5 minutes
- Extraction of formazan: after the MTT incubation period, the tissues were rinsed three times with DPBS and allowed to dry. The tissues were transferred into 12-well plates and immersed in 2 mL isopropanol, sealed to inhibit evaporation. Extraction was carried out protected from light at room temperature either overnight or, alternatively, at least 2 hours with shaking on a plate shaker.
Before using the extracts, the plate was shaken for at least 15 minutes on a plate shaker and the inserts were pierced with an injection needle. The extract was pipetted up and down 3 times before 2 x 200 µL aliquots per each tissue were transferred into a 96-well plate. Optical density (OD) was measured with a filter band without reference wavelength in a plate spectrophotometer using isopropanol as a blank.
- Wavelength: 570 nm
- Filter bandwidth: maximum ± 30 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: tissues pass analysis for tissue viability
- Barrier function: tissues pass analysis for tissue functionality
- Morphology: presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers.
- Contamination: absence of bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture) as well as absence of HIV1-virus, Hepatitis B virus and Hepatitis C virus
Please also refer to the field "Attached background material" below.

PREDICTION MODEL / DECISION CRITERIA
The mean optical density (OD) of the three negative control tissues was calculated after blank correction. This value corresponds to 100 % tissue viability in the current test. For each individual tissue treated with the test item or the positive control, the individual relative tissue viability was calculated according to the following formula:
Relative viability (%) = [mean ODtest item / positive control / ODmean of negative control] * 100
For the test item and the positive control the mean relative viability ± relative standard deviation of the three individual tissues was calculated and used for classification according to the following prediction model:
Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with DPBS. The test item is considered to be irritant to skin in accordance with regulation EC 1272/2008 (UN GHS “Category 2”), if the tissue viability after exposure and post-incubation is less or equal to 50%. The test substance may be considered as non-irritant to skin in accordance with regulation EC 1272/2008 and UN GHS “No Category” if the tissue viability after exposure and post-treatment incubation is more than 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 (39 mg/cm²) of the test item
The test item was applied directly atop the EpiDermTM tissue using an application spoon avoiding compression of the test item. The test item was spread to match size of the tissue by gently shaking the inserts or by using a bulb-headed Pasteur pipette.

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL of the vehicle

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of 5 % SDS solution
Duration of treatment / exposure:
60 ± 1 minutes
Duration of post-treatment incubation (if applicable):
approx. 42 hours
Number of replicates:
triplicates
Irritation / corrosion parameter:
% tissue viability
Remarks:
(mean)
Value:
85
Vehicle controls valid:
not examined
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, no additional controls were necessary.
- Colour interference with MTT: mixture of 25 mg of the test item per 300 µL aqua dest. and per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, no additional controls were necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (value: 1.500).
- Acceptance criteria met for positive control: mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3.9 %)
- Acceptance criteria met for variability between replicate measurements: standard deviation of relative tissue viability of replicate tissues of all dose groups was ≤ 18% (0.4 % - 16.7 %).
Please also refer to the field "An other information on results incl. tables" below.

Table 1: Results of the test item zinc 5-nitroisophthalate

Name

Negative Control

Positive Control

Test item

Tissue

1

2

3

1

2

3

1

2

3

absolute OD570

1.635

1.509

1.498

0.106

0.096

0.102

1.585

1.142

1.175

1.595

1.511

1.510

0.110

0.098

0.102

1.630

1.205

1.171

OD570

(blank-corrected)

1.592

1.466

1.455

0.062

0.053

0.059

1.542

1.098

1.132

1.552

1.468

1.467

0.066

0.055

0.059

1.587

1.162

1.127

mean OD570of the duplicates (blank-corrected)

1.572

1.467

1.461

0.064

0.054

0.059

1.564

1.130

1.129

total mean OD570of 3 replicate tissues (blank-corrected)

1.500*

0.059

1.275

SD OD570

0.062

0.005

0.251

relative tissue viability [%]

104.8

97.8

97.4

4.3

3.6

3.9

104.3

75.4

75.3

mean relative tissue viability [%]

100.0

3.9**

85.0

SD tissue viability [%]***

4.2

0.4

16.7

CV [% viabilities]

4.2

8.9

19.7

* Blank-corrected mean OD570 nmof the negative control corresponds to 100% absolute tissue viability.

** Mean relative tissue viability of the three positive control tissues is  20%.

*** Standard deviation (SD) obtained from the three concurrently tested tissues is  18%

Table 2: Historical data

 

Mean

OD570±30nm

Negative control (NK)

Mean

Relative Viability [%] Positive control (PC)

SD Viability [%]

Mean

1.869

4.1

4.2

SD

0.348

2.0

4.8

n

34

34

121

Historical data were generated from 2009 to 2017.

Interpretation of results:
GHS criteria not met
Conclusions:
The test item is not irritating to the skin.
According to the Regulation (EC) No 1272/2008 and subsequent regulations, the test item is not irritating to the skin.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
1981-05-12
Deviations:
yes
Remarks:
no detailed description of test performance, no extended observation to determine reversibility
GLP compliance:
no
Remarks:
not required at time of the study
Test material information:
Composition 1
Species:
rabbit
Strain:
Vienna White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: M. Gaukler, Offenbach, Germany
- Weight (mean): 2.8 kg
- Diet: ssniff K-Standarddiät für Kaninchen (INTERMAST GmbH, Soest, Germany)
- Water: ad libitum
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approximately 50 mg test item
Duration of treatment / exposure:
not applicable
Observation period (in vivo):
24, 48 and 72 hours
Number of animals or in vitro replicates:
6 rabbits (4 males / 2 females)
Details on study design:
STUDY DESIGN:
The test substance was instilled into the conjunctival sac of the right eye of each rabbit. The upper and lower lids were then gently hold together for about 1 second before releasing. The left eye remaind as untreated control.

SCORING SYSTEM:
According to Draize scale.

Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
not fully reversible within: 72 h
Remarks on result:
other: no follow-up after 72 h
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
not fully reversible within: 72 h
Remarks on result:
other: no follow-up after 72 h
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #3
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
not fully reversible within: 72 h
Remarks on result:
other: no follw-up after 72 h
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #4
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #4
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #4
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
not fully reversible within: 72 h
Remarks on result:
other: no follow-up after 72 h
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #4
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #5
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #5
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #5
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
not fully reversible within: 72 h
Remarks on result:
other: no follow-up after 72 h
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #5
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #6
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #6
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #6
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
not fully reversible within: 72 h
Remarks on result:
other: no follow-up after 72 h
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #6
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritant / corrosive response data:
In addition, the eye secretion was examined. At the 24 hour observation, 2/6 animals showed eye secretion, which is slight above normale secretion.

Interpretation of results:
GHS criteria not met
Conclusions:
The substance is not irritating to the eyes.
According to the EC Regulation No. 1272/2008 and subsequent adaptations, the substance is not classified as irritating to the eyes.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Skin irritation:

The substance was not observed to be irritating to the skin in a reliable in vitro skin irritation study according to OECD 439.

Eye irritation:

The substance was not observed to be irritating to the eyes in a reliable in vivo eye irritation study equivalent or similar to OECD 405.

Justification for classification or non-classification

Skin irritation:

The substance does not possess a skin irritating potential based on an in vitro OECD 439 (2015) test and does not require classification as skin irritant according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

Eye irritation:

The substance does not possess an eye irritating potential based on an in vivo OECD 405 (1981) test and does not require classification as eye irritant according to Regulation (EC) No 1272/2008 and its subsequent adaptations.