Registration Dossier

Administrative data

Description of key information

Testing strategy Analysis before performing in vivo study 

The sequential testing strategy was used as it is recommended in supplement to TG 405(2012).

SAR systems are not applicable to this type of test substance.

Validated and accepted in vitro tests for eye corrosion/irritation and in vitro skin irritation have been conducted prior to decide performing the in vivo test. It was BCOP test (OECD TG No. 437), In vitro Skin Irritation Test (OECD TG No. 439) and In vitro Eye Irritation Test (OECD TG No.492). The results did not allow the classification of test substance with regard to its potential to cause eye irritation or serious eye damage. QSAR model is not applicable to this type of test substance.

- Study No. 392/16/4AI: Direct Brown 103 - In vitro Skin Irritation Test (EpiDermTM Model); VUOS-CETA Report No. 17-255, 2017.

Result: no category in regard to skin irritation

- Study No. 392/16/5BCOP: Direct Brown 103 - Bovine Corneal Opacity and Permeability Test; VUOS-CETA Report No. 16-505, 2016

Result: no prediction can be made

- Study No. 392/16/5AI: Direct Brown 103 - In vitro Eye Irritation Test (EpiOcularTM Model); VUOS-CETA Report No. 17-361, 2017.

Result: substance potentially requiring classification and labelling, further testing with other test methods will be required

Therefore it was decided to perform in vivo eye irritation study

Study No.: 392/16/5: Direct Brown 103 - Acute Eye Irritation/Corrosion; VUOS-CETA Report No. 17 -645, 2017.

Result:the test substance, Direct Brown 103, is not irritating to the eye of rabbit. The initial irritation (1 hour after exposure) is caused by mechanical irritation of petty particles of the test substance.

The test substance Direct Brown 103 was non-irritating based on in vitro test and based on in vitro eye irritation tests and in vivo test on rabbit eye it is not irritating to the eye. Therefore conclusion is non-irritating for skin and eye.

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:
6.1.2017 - 20.1.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Adopted: 28th July, 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Commission Regulation (EC) No. 761/2009, 23rd July 2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: tissue for research puposes from accredited institutions
Source strain:
other: Keratinocyte strain 00267
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUEthe reconstructed human epidermal model EpiDerm (EPI-200 ver. 2.0, MatTek, Bratislava, Slovakia); Lot No. 23388, kit ETEMPERATURE USED FOR TEST SYSTEMculture conditions 37±1°C, 5±1 % CO2, moistened tissueREMOVAL OF TEST MATERIAL AND CONTROLS- Volume and number of washing steps: thoroughly rinsed with PBS- Observable damage in the tissue due to washing: After rinsing, tissues remained uneven coloured brown. The tissue with the lowest OD570 was pierced at rinsing what could have influence on lower intensity of its colouring at MTT test.MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE- MTT concentration: 1 mg·mL-1- Incubation time: 185 min- Spectrophotometer: Libra S22 at 570 nm. Isopropyl alcohol serves as a blank. Allowed band width is 2-3 nm. No external filter was used.FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATABased on Certificate of Analysis the model passed all parametres for viability, barrier function, sterility.NUMBER OF REPLICATE TISSUES: 3NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:1. Direct MTT reduction - functional check in tubes2. Colour interference 3. MTT testPREDICTION MODEL / DECISION CRITERIAOECD Test Guideline No. 439 (1), par. 36:- In case the test chemical is found to be non-corrosive (e.g., based on TG 430, 431 or 435), and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%, the test chemical is considered to be irritant to skin in accordance with UN GHS (3) Category 2. - The test chemical may be considered as non-irritant to skin in accordance with 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:
The test substance (25 mg of substance/surface ratio 39.7 mg/cm2) was placed directly on tissue moistened with 25 µL of PBS. The material was spread on the tissue surface.NC: sterile PBS (phosphate buffered saline) MatTek 092316MHEPC: 5 % SDS (sodium dodecyl sulphate), MatTek, Lot No. 111516ZSA
Duration of treatment / exposure:
60±1 minutes (25 minutes at room temperature and the remaining 35 minutes at culture conditions)
Duration of post-treatment incubation (if applicable):
After 24±2 hours incubation period, the medium is replaced by fresh one. Tissues are incubated for another 18±2 hours (post-treatment incubation).
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
60.9
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
After rinsing, tissues remained uneven coloured brown. The tissue with the lowest OD570was pierced at rinsing what could have influence on lower intensity of its colouring at MTT test.The mean OD570 of the NC tissue was 2.004 ±0.047 which meets the acceptance criteria of ≥ 0.8 and ≤ 2.8.The mean viability of the PC tissues expressed as % of the negative control tissues is 2.4% which meets the acceptance criterion of ≤ 20 %. The SD calculated from individual % tissue viabilities of the 3 identically treated replicates for the positive control, negative control and test substance was 12.4 what is < 18 %.All study acceptance criteria were fulfilled.

Direct MTT reduction: functional check in tubes

25 mg ofthe test substance was addedto 1.0 mL of MTT medium. Suspension was incubated for 1 hour at culture conditions. After incubation, the medium was coloured red-brown. The test substance does not reduce MTT directly.

Colour interference

The test substance is soluble in water for injection. OD570 of solution in water for injection was > 3 what is > 0.08.

The test substance is not soluble in isopropyl alcohol. Average OD570 value from 2 wells was 0.005 what is < 0.08.

It means that the test substance will go well washed from the tissue and any residue is not dissolved in isopropyl alcohol. On the basis of results obtained, it was decided do not use concurrent colorant control in the MTT test.

MTT test:

OD570values obtained at the MTT test, their averages, standard deviations (%) and relative viabilities

 

Treatment

OD570

Avg

SD

Average viability

 

1

2

3

 

 

(% NC)

NC

 

PBS

2.022

1.940

2.050

2.004

0.047

100.0 

%

100.9

96.8

102.3

100.0

2.33

C3

 

392/16

1.366

1.427

0.871

1.221

0.249

60.9 

%

68.2

71.2

43.5

60.9

12.42

PC

 

5% SDS

0.044

0.051

0.050

0.048

0.003

2.4

%

2.2

2.5

2.5

2.4

0.15

Interpretation of results:
GHS criteria not met
Conclusions:
Under the above-described experimental design, average viability of tissues treated by the test substance Direct Brown 103 was 60.9 % of negative control average value, i.e. viability was > 50 %. The effect of the test item was negative in EpiDermTM model (tissues were not damaged).According to the classification criteria, the test substance, Direct Brown 103, is considered to have no category in accordance with UN GHS and is therefore considered a non-irritant.
Executive summary:

The test item, Direct Brown 103, was assayed for the in vitro skin irritation in human epidermal model EpiDermTM. The test was performed according to the OECD Test Guideline No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method test (2015) and Protocol for: In Vitro EpiDermTMSkin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT.

In the preliminary experiments neither colour interference with the endpoint nor direct MTT reduction were found.

After pre-incubation of tissues, 25 mg of the test substance was placed directly atop to the previously moistened tissue and it was spread on the entire tissue surface. The length of exposure was 60 minutes. Three tissues were used for the test substance and for positive and negative controls.

After removal of the test substance, tissues were post-incubated for approximately 42 hours. Three hours incubation with MTT and two hours extraction period with shaking followed then. Optical density (OD570) of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

Under the above-decribed experimental design, average viability of tissues treated by the test substance was 60.9 %, i.e. viability was > 50 %.

The effect of the test substance was negative in EpiDermTMmodel (tissues were not damaged).

According to the classification criteria, the test substance, Direct Brown 103, is considered to have no category in regard to skin irritation.

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:
07.09. – 08. 09. 2016
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES- Source: Breeding service CHOVSERVIS a.s., division TORO® Hlavečník, Hradec Králové, Czech Republic- Characteristics of donor animals (e.g. age, sex, weight): The eyes were enucleated as soon as possible after death. No detergent was used. Only healthy animals (12 to 60 months old) considered suitable for entry into the human food chain were used as a source of corneas for use in the BCOP test. - Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):The risk of contamination was minimized (e.g., by keeping the container containing the eyes on ice, by adding antibiotics to the HBSS used to store the eyes during transport (e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL).- Time interval prior to initiating testing: The time interval between collection of the eyes and use of corneas in the BCOP was minimized (typically collected and used on the same day). All eyes used in the assay were from the same group of eyes collected on a specific day.- indication of any existing defects or lesions in ocular tissue samples: Only corneas from eyes free of defects including scratched, and neovascularisation were used. The isolated corneas, after achieve normal metabolic activity (inductive incubation at 32 ± 1°C for one hour), were examined again. The corneas that show macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or a baseline opacity >7 opacity units were discarded.
Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL - Amount(s) applied (volume or weight with unit): 750 μl of suspension2g of the test substance was suspended in 10 mL of 0.9% sodium chloride solution.
Duration of treatment / exposure:
4 hrs
Duration of post- treatment incubation (in vitro):
1.5 hr
Number of animals or in vitro replicates:
The results were based on the selection criteria for the eyes, as well as the positive and negative control responses.Number of corneas per group: Exposed group (test substance) - 3 corneas (No. 8, 9, 11) Positive control group (20% Imidazole in 0.9% NaCl) – 3 corneas (No. 12, 13, 14) Negative control group (0.9% NaCl) – 3 corneas (No. 2, 3, 5)
Details on study design:
SELECTION AND PREPARATION OF CORNEASSelection criteria for eyes used in BCOP: Only corneas from eyes free of defects including scratched, and neovascularisation were used. The isolated corneas, after achieve normal metabolic activity (inductive incubation at 32 ± 1°C for one hour), were examined again. The corneas that show macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded. Preparation: Corneas free of defects were dissected with a 2 to 3 mm rim of sclera remaining to assist in subsequent handling, with care taken to avoid damage to the corneal epithelium an endothelium. Isolated corneas were mounted in specially designed corneal holders that consisted of anterior and posterior compartments, which interfaced with the epithelial and endothelial sides of the cornea, respectively. Both chambers were filled to excess with pre-warmed Eagle's Minimum Essential Medium (EMEM). The device was then equilibrated at 32 ± 1°C for at least one hour in water bath to allow the corneas to equilibrate with the medium and to achieve normal metabolic activity, to the extent possible. Following the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Any corneas that showed macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded. Each test group (test substance, concurrent negative and positive controls) consisted of the three eyes. The three corneas with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment and positive control groups. QUALITY CHECK OF THE ISOLATED CORNEASFrom 22 eyes the 5 eyes were eliminated after inductive incubation, because the baseline opacity values were >7. Nine corneas were used for the study (the corneas No. 2, 3, 5, 8, 9, 11, 12, 13 and 14), 4 eyes was superfluous and the remaining 3 eyes were used for the testing of another substance.NUMBER OF REPLICATESNumber of corneas per group:Exposed group (test substance) - 3 corneas (No. 8, 9, 11) Positive control group (20% Imidazole in 0.9% NaCl) – 3 corneas (No. 12, 13, 14) Negative control group (0.9% NaCl) – 3 corneas (No. 2, 3, 5) NEGATIVE CONTROL USED0.9% NaClSOLVENT CONTROL USED (if applicable)0.9% NaClPOSITIVE CONTROL USED20% ImidazoleAPPLICATION DOSE AND EXPOSURE TIME750 µL of application form (2g of the test substance was suspended in 10 mL of 0.9% sodium chloride solution). 4 hrsTREATMENT METHOD: closed-chamber method POST-INCUBATION PERIOD: REMOVAL OF TEST SUBSTANCE- Number of washing steps after exposure period: - POST-EXPOSURE INCUBATION: After the exposure period, the negative control and the positive control substance was removed from the anterior chamber with EMEM (containing phenol red - the effectiveness of rinsing acidic or alkaline materials). The corneas were given a final rinse with EMEM (without phenol red). The EMEM (without phenol red) was used as a final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded.The test substance was removed from the anterior chamber with EMEM (containing phenol red) and sequentially was removed mechanically using a cotton swab – very gently (the test substance was coloured and had tendency to stick on the cornea surface) ; also the test substance was removed from the anterior chamber with EMEM (containing phenol red) once more. The corneas (applied the test substance) were also repeatedly rinsed with EMEM (without phenol red). However the test substance was not completely removed. The corneas stayed mild coloured by the test substance. The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded.METHODS FOR MEASURED ENDPOINTS: - Corneal opacity:measured quantitatively with the aid of an opacitometer (Opacitometer, MC2 - Le spécialiste du laboratoire – France) resulting in opacity values measured on a continuous scale- Corneal permeability: The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured with the aid of UV/VIS spectrophotometry (Spectrophotometer GENESYSTM 10 UV/VIS Scanning). The values of absorbance measured at 490 nm were recorded as optical density (OD490) values. This term was used because the measuring is performed with visible light spectrophotometer using a standard 1 cm path length.SCORING SYSTEM: In Vitro Irritancy Score (IVIS)IVIS = mean opacity value + (15 x mean permeability OD490 value)DECISION CRITERIA: IVISUN GHS ≤ 3 No Category> 3; ≤ 55 No prediction can be made ≥ 55 Category 1
Irritation parameter:
in vitro irritation score
Value:
33.95
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects:
After exposure the test substance was removed from the corneas. In spite of this procedure the corneas treated by the test substance remained coloured. This colouring of the corneas had influence on the measuring of opacity what could result in higher opacity values.
Interpretation of results:
other: no prediction can be made
Conclusions:
The In Vitro Irritancy Score (IVIS) for Direct Brown 103 was 33.95. On the basis of score (IVIS) given above the classification according to the criteria of the UN GHS could not be performed.No prediction can be made on test substance potential to cause eye irritation or serious eye damage.
Executive summary:

The test substance, Direct Brown 103, was tested for the evaluation the potential ocular corrosivity or severe irritancy as measured by its ability to induce opacity and increased permeability in an isolated bovine cornea.

The test was performed according to the OECD Test Guideline No. 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, Adopted 26th July 2013.

 

The test was performed using nine isolated bovine corneas. The testing was performed on three groups of corneas: test substance treatment group, positive control group and negative control group. Three corneas per group were used.

Closed-chamber method was used, because the test substance was applicable by micropipette. The opacity and permeability of each cornea were measured. The In Vitro Irritancy Score (IVIS) was calculated from the values of opacity and permeability.

 

The In Vitro Irritancy Score (IVIS) for Direct Brown 103 was 33.95.

On the basis of score (IVIS) given above the classification according to the criteria of the UN GHS could not be performed.

No prediction can be made on test substance potential to cause 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:
19.4.2016 - 21.4.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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)
Version / remarks:
Adopted: 28 July 2015
Deviations:
yes
Remarks:
see Any other information ...
GLP compliance:
yes (incl. QA statement)
Species:
human
Strain:
other: Keratinocyte strain 4F1188
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicabilityCell damage (cytotoxicity), playing an important, if not the primary, mechanistic role in determining the overall serious eye damage/eye irritation response of a chemical regardless of the physicochemical processes underlying tissue damage, is followed in this test.This test uses an in vitro procedure allowing the identification of chemicals not requiring classification and labelling for eye irritation or serious eye damage in accordance with UN GHS. This test is not able to distiguih between serious eye damage and eye irritation. - Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell liveThe RhCE tissues are reconstructed from primary human cells, which have been cultured for several days to form a stratified, highly differentiated squamous epithelium, morphologically similar to that found in the human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.The developer/supplier provided demonstration that each batch of the RhE model used meets acceptable production release criteria (Tissue certificate). The reconstructed human cornea-like epithelial model EpiOcular™ comes from MatTek, Bratislava, SK, supplied with Certificate of Analysis. Lots No. of tissues used for this test: 23776 kit AOn the day of receipt, EpiOcular tissues are conditioned to release transport stress related compounds and debris by incubation in assay medium delivered by MatTek for test performance for 1 hour at standard culture conditions and, after media replacement, overnight (following 16-24 hours) also standard at culture conditions.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The test substance (50 mg of substance/surface ratio 39.7 mg/cm2)/per tissue is placed directly atop to the tissue moistened with 20 µL of PBS. The material should cover the entire tissue surface.
Duration of treatment / exposure:
6 ± 0.25 hours
Duration of post- treatment incubation (in vitro):
18±0.25 hours
Number of animals or in vitro replicates:
3
Details on study design:
- Details of the test procedure used1. Direct MTT reduction - functional check in tubesThe test was performed as a part of Study No. 392/16/4AI: Direct Brown 103 - In vitro Skin Irritation Test (EpiDermTM Model); VUOS-CETA Report No. 17-255, 2017. The test substance does not reduce MTT directly.2. Colour interference (NSCC)The test was performed as a part of Study No. 392/16/4AI: Direct Brown 103 - In vitro Skin Irritation Test (EpiDermTM Model); VUOS-CETA Report No. 17-255, 2017. The test substance does not interfere with the endpoint.3. MTT testA single testing, composed of 3 replicate tissues, is run (plus 3 for the positive control (PC) and 3 for the negative control (NC)).After exposure time: extensive rinsing the tissues with PBS brought to room temperature- RhCE tissue construct used, including batch numberThe reconstructed human cornea-like epithelial model EpiOcular™ comes from MatTek, Bratislava, SK. Lots No. of tissues used for this test: 23776 kit A.- Doses of test chemical and control substances usedThe test substance (50 mg of substance/surface ratio 39.7 mg/cm2) is placed directly atop to the tissue moistened with 20 µL of PBS. The material should cover the entire tissue surface.PC: Methyl Acetate, MatTek, Lot No. 032817ISA, exp. 28/03/2018NC:water for injection- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)6 ± 0.25 hours at standard culture conditions (37±1°C, 5±1 % CO2, humidified incubator)25 ± 2 minute immersion incubation (post-soak) at room temperature18 ± 0.25 hours at standard culture conditions (37±1°C, 5±1 % CO2, humidified incubator)- Description of any modifications to the test procedureSee Any other information ...(Deviations) - Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)OD570 is measured on a plate reader Biotek Epoch. Isopropyl alcohol serves as a blank. No external filter is used.- Description of the method used to quantify MTT formazanOptical density (OD570) of isopropyl alcohol extracts was measured on a plate reader. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction modelThe percentage tissue viability cut-off value distinguishing classified from non-classified test chemicals is 60%. Results should thus be interpreted as follows:- The test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%. In this case no further testing in other test methods is required.- The test chemical is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post exposure incubation is less than or equal (≤) to 60%. - Positive and negative control means and acceptance ranges based on historical data1) The negative control OD > 0.8 and < 2.5, 2) The mean relative viability of the positive control is below 50% of control viability 3) The difference of viability between the two relating tissues of a single chemical is < 20% in the same run. This applies also to the killed controls and the colorant controls which are calculated as percent values related to the viability of the relating negative control.
Irritation parameter:
other: average viability
Run / experiment:
1
Value:
18.7
Negative controls validity:
valid
Positive controls validity:
not valid
Remarks:
See Any other information ...
Remarks on result:
positive indication of irritation
Other effects:
- Effects of rinsing or washing:After washing of the test substance off tissues, tissues remained coloured brown. Tissues remaind coloured brown even after extraction with isopropyl alcohol

Table 1: OD570values obtained at the MTT test, their averages, standard deviations (%) and relative viabilities

 

Treatment

OD570of tissues

avg/

avg[%]

SD/

SD[%]

Average viability

 

Tissue 1

Tissue 2

Tissue 3

(% NC)

NC

water

1.522

1.261

1.558

1.447

0.132

100.0

 

% NC

105.21

87.17

107.70

100.0

9.2

C3

392/16

0.279

0.267

0.269

0.272

0.004

18.7

 

% NC

19.29

18.46

18.59

18.7

1.9

PC

99% MA

0.858

0.309

0.247

0.471

0.274

32.6

 

% NC

59.31

21.36

17.07

32.6

58.2

The data presented are corrected by subtraction of OD570isopropyl alcohol itself (blank, 0.039).

Acceptance criteria compliance

1) The negative control OD > 0.8 and < 2.5 (1.447)

2) The mean relative viability of the positive control is below 50% of control viability (32.6 %)

3) The difference of viability between the three relating tissues of a single chemical is < 20% in the same run. This applies also to the killed controls and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

The difference of viability between the three relating tissues of the negative control was 9.2%. The difference of viability between the three relating tissues of the test substance was 6.2% what is < 20%. This criterion was fulfilled.

The difference of viability between the positive control tissues of was > 20%. This criterion was not fulfilled. For solution see Deviations.

Deviations

Three tissues were used for the test substance, three for negative and three for positive control – a deviation from study plan.

This deviation had no impact on the outcome of study.

The difference of viability between the positive control tissues of was > 20%. Average viability was 32.6% what is < 50%.

This deviation had no impact on the outcome of study.

Interpretation of results:
study cannot be used for classification
Conclusions:
Under the above-described experimental design, average viability of tissues treated by the test substance Direct Brown 103 was 18.7 % of negative control average value. The effect of the test substance was positive in EpiOcularTM model (tissues were damaged). According to the classification criteria given in chapter 4.5., the test substance, Direct Brown 103, is identified as substance potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1). Further testing with other test methods will be required.
Executive summary:

The test substance, Direct Brown 103, was assayed for the in vitro eye irritation in human epidermal model EpiOcularTM. The test was performed according to the OECD Test Guideline No. 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage. Details of the procedure are given in Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals (MatTek 06/29/2015).

After pre-incubation and wetting of tissues, 50 mg of the test substance was placed directly atop to the tissue and it was spread on the entire tissue surface. Length of exposition was 6 hours at 37±1°C in humidified CO2 incubator (5±1% CO2). Two tissues were used for the test substance and every control. Two tissues more were used as colorant control to correction of possible colour interference, which was undergo the entire testing procedure excepting of incubation with MTT medium.

After removal of the test substance, tissues were post-soaked in medium for approximately 25 minutes and post-incubated for about 18 hours at culture conditions. Three hours incubation with MTT and two hours extraction period with shaking followed then. Optical density (OD570) of isopropyl alcohol extracts was measured on a plate reader. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

Tests for colour interference and direct reduction were performed as a part of another study. Direct reduction neither colour interference were not found.

Under the above-described experimental design average viability of treated tissues was 18.7% i.e. viability was ≤ 60 %.

The effect of the test substance was positive in EpiOcularTMmodel (tissues were damaged).

According to the classification criteria given in chapter 4.5., the test substance, Direct Brown 103, is identified as substance potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Further testing with other test methods will be required.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11.09. – 18.09.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
Commission Regulation (EU) 2017/735,Published in O.J.L. 112, 2017
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
Adopted October 2, 2012
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
FORM AS APPLIED IN THE TEST (if different from that of starting material)granular powder, so it was grounded to a fine dust
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS- Source: breeding farm VELAZ s.r.o., Lysolaje, Czech Republic, RČH CZ 21760118- Weight at study initiation: 2.70 – 3.00 kg- Housing: conventional animal room – individually in metallic cages- Diet (e.g. ad libitum): ad libitum- Water (e.g. ad libitum): ad libitum- Acclimation period: 5 days, no signs of disease were observedENVIRONMENTAL CONDITIONS- Temperature (°C): 20 ± 3°C, permanently monitored- Humidity (%): 30 – 70%, permanently monitored- Photoperiod (hrs dark / hrs light): 12 h/12 h
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
A dose of 0.1g of the test substance was applied to the test site.
Duration of treatment / exposure:
The test substance was placed into the conjunctival sac of one eye of animals after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second in order to prevent loss of the substance. The other eye, which was untreated, serves as a control.The substance was solid and has not been removed from the eye of the test animal by physiological mechanisms at the first observation time point of 1 hour after treatment. The eye was rinsed with physiological saline solution.
Observation period (in vivo):
examined at 1, 24, 48 and 72 hours after application
Number of animals or in vitro replicates:
3 animals
Details on study design:
REMOVAL OF TEST SUBSTANCE - Washing (if done): rinsed with physiological saline solution - Time after start of exposure: 1h SCORING SYSTEM: To the ocular reactions observed at each time interval the grades were assigned according to the grading system given in Method B.5 – Acute Eye Irritation/Corrosion, Commission Regulation (EU) 2017/735, Published in O.J.L. 112, 2017TOOL USED TO ASSESS SCORE: After recording the observations at 72 hours, the eyes of rabbit were examined with the hand slit-lamp.
Irritation parameter:
cornea opacity score
Basis:
animal: 7
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal: 9
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal: 11
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal: 7
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal: 9
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal: 11
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
animal: 7
Time point:
24 h
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 24h
Irritation parameter:
conjunctivae score
Basis:
animal: 9
Time point:
24 h
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 24 h
Irritation parameter:
conjunctivae score
Basis:
animal: 11
Time point:
24 h
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 24 h
Irritation parameter:
chemosis score
Basis:
animal: 7
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
chemosis score
Basis:
animal: 9
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
chemosis score
Basis:
animal: 11
Time point:
24 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 24 h

No alterations of control eyes were observed during the whole study.

Interpretation of results:
GHS criteria not met
Conclusions:
The following changes were observed on eye at 1 hour after application: conjunctivae – diffuse, crimson colour, individual vessels not easily discernible, and chemosis – obvious swelling with partial eversion of lids was observed in all rabbits. Some hyperaemic blood vessels of conjunctivae were observed in all rabbits at 24 hours after application. Some swelling above normal was observed in rabbit No. 11 at 24 hours after application. No alterations were observed in all rabbits at 48 and 72 hours after application. All eye lesions disappeared at least at 48 hours after treatment. Evaluation of results after single application demonstrated that the test substance, Direct Brown 103, is not irritating to the eye of rabbit. The initial irritation (1 hour after exposure) is caused by mechanical irritation of petty particles of the test substance.
Executive summary:

The test substance, Direct Brown 103, was tested for the assessment of eye irritation/corrosion effects using albino rabbit (New Zealand Albino breed).

 

The test was performed according the following documents:

Method B.5 – Acute Eye Irritation/Corrosion, Commission Regulation (EU) 2017/735, Published in O.J.L. 112, 2017

OECD Test Guideline No. 405 Acute Eye Irritation/Corrosion. Adopted October 2012.

Before in vivo testing the sequential testing strategy as it is recommended in supplement to TG 405(2012) was respected.

The test was performed initially using one animal (No. 7). Because no corrosive or severe irritating effects were observed in initial test, the response was confirmed using two additional animals (No. 9 and No. 11).

The following changes were observed on eye at 1 hour after application: conjunctivae – diffuse, crimson colour, individual vessels not easily discernible, and chemosis – obvious swelling with partial eversion of lids was observed in all rabbits.

Some hyperaemic blood vessels of conjunctivae were observed in all rabbits at 24 hours after application. Some swelling above normal was observed in rabbit No. 11 at 24 hours after application.

No alterations were observed in all rabbits at 48 and 72 hours after application. All eye lesions disappeared at least at 48 hours after treatment.

No clinical signs of systemic intoxication were detected.

Evaluation of results after single application demonstrated that the test substance, Direct Brown 103, is not irritating to the eye of rabbit. The initial irritation (1 hour after exposure) is caused by mechanical irritation effect of test substance petty particles.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

The test substance Direct Brown 103 was non-irritating based on in vitro test and based on in vitro eye irritation tests and in vivo test on rabbit eye is not irritating to the eye. Therefore conclusion is non-irritating for skin and eye.