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Administrative data

Description of key information

In an in vitro EpiDerm assay conducted in accordance with OECD Test Guideline 439 and to GLP, "Karstedt concentrate" was non-cytotoxic and, hence, non-irritant to skin (Spruth, 2016a).

In an in vitro bovine corneal opacity and permeability (BCOP) assay conducted in accordance with OECD Test Guideline 437, and to GLP, "Karstedt concentrate" did not induce a response indicative of a corrosive or severe irritant on the eye (Spruth, 2016b). As the in vitro BCOP test is considered applicable for ‘Karstedt concentrate’ and the negative results obtained are adequate to conclude that no classification is needed, an in vivo eye irritation study is not necessary.

No relevant respiratory tract irritation data were identified.

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:
21 April - 27 May 2016
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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
other:
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDermTM (EPI-200, Lot no. 23338) MatTek In Vitro Life Science Laboratories, s.r.o, Mlynské Nivy 73, 821 05 Bratislava II, Slovak Republic
Source strain:
other: Reconstructed human epidermis model (see details below)
Details on animal used as source of test system:
Not applicable
Justification for test system used:
The reconstructed human epidermis model system is suitable to test solids, liquids, semi-solids and waxes. The liquids may be aqueous or non-aqueous; solids may be soluble or insoluble in water.
Vehicle:
unchanged (no vehicle)
Details on test system:
EpiDerm is a three-dimensional reconstructed human epidermis model, comprised of normal, human-derived epidermal keratinocytes, which had been cultured to form a multilayered, highly differentiated model of the human epidermis. It consisted of organised basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.

The principle of the reconstructed human epidermis model test is based on the premise that irritant substances are able to penetrate the stratum corneum by diffusion and are cytotoxic to the cells in the underlying layers. Cell viability was measured by dehydrogenase conversion of the vital dye MTT (3-[4,5- Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue), into a blue formazan salt that was quantitatively measured after extraction from tissues. Irritant substances were identified by their ability to decrease cell viability below defined threshold levels (i.e.≤ 50% for UN GHS category 2 irritants).

The EpiDerm™ System was manufactured according to defined quality assurance procedures. All biological components of the epidermis and the culture medium were tested by the manufacturer for viral, bacterial, fungal and mycoplasma contamination. MatTek corporation determines the ET50 value following exposure to Triton X-100 (1%) for each EpiDerm lot. The ET50 must fall within a range established based on a historical database of results.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL of undiluted test item were applied to the skin model with a surface area of 0.63 cm2 to uniformly cover the skin surface. Three replicate tissues were employed. At the end of the exposure period, the test item was carefully washed from the skin surface with D-PBS.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL D-PBS was added to each of the three negative control skin units.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL SDS was added to each of the three positive control skin units.
- Concentration (if solution): 5% aqueous solution
Duration of treatment / exposure:
Exposure for 60 minutes: 35 minutes at 37°C, 5% CO2 and 95% humidity followed by 25 minutes at room temperature.
Duration of post-treatment incubation (if applicable):
The post-treatment incubation period of the rinsed tissues in fresh assay medium was 42 hours.
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Remarks:
Score is a percentage (%) of negative control.
Run / experiment:
Mean
Value:
90.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
The mean OD of the NC of 3 tissues was 1.773 and was well within the acceptable range of ≥ 1.0 to ≤ 2.5.
Positive controls validity:
valid
Remarks:
The viability of cells treated with the PC was 6.0% of the NC and fulfilled the acceptance criterion of ≤ 20%.
Remarks on result:
no indication of irritation
Remarks:
If the mean relative viability <=50% the test substance is considered to be irritant to skin.
Other effects / acceptance of results:
The standard deviation of all triplicates determined was below the limit of acceptance of 18%. Hence, all acceptance criteria required were fulfilled.

No discolorations were noted in the test for colour change under aqueous conditions. Additionally, no change of colour was noted in the test for MTT interference potential. Therefore the test item did not interact with the MTT measurement and no additional test had to be performed.

 

The summary of the OD results is given below:

   Optical density (n=3 tissues)  SD  % OD540 compared to the control
 Negative control (D-PBS)  1.773  0.0907  -
 "Karstedt concentrate"  1.607  0.091  90.6
 Positive control (5% SDS)  0.106  0.012  6.0
Interpretation of results:
GHS criteria not met
Conclusions:
In an in vitro EpiDerm assay conducted in accordance with OECD Test Guideline 439 and to GLP, "Karstedt concentrate" was non-cytotoxic and, hence, non-irritant to skin.
Executive summary:

“Karstedt concentrate” was tested for skin irritation potential in an in vitro EpiDerm assay, using reconstructed human epidermis, conducted in accordance with OECD Test Guideline 439 and to GLP. The mean cell viability following exposure to the test substance was calculated to be greater than 50% (90.6% of the negative controls), and it was therefore considered to be non-cytotoxic and predicted to be non-irritant to skin. The positive and negative controls were considered valid.

Under the conditions of this assay, “Karstedt concentrate” would not be classified as "irritant" according to EU CLP criteria (EC 1272/2008).

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:
21-27 April 2016
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)
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:
published in the Official Journal of the European Union L324, dated December 09, 2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: Bovine eyes from cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Source of eyes: 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 (HBSS) containing 1% Penicillin/Streptomycin.
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): 0.75 mL
Duration of treatment / exposure:
10 minutes
Observation period (in vivo):
Not applicable
Duration of post- treatment incubation (in vitro):
2 hours
Number of animals or in vitro replicates:
Not applicable
Details on study design:
SELECTION AND PREPARATION OF CORNEAS: Only corneas from eyes free of defects were used. The corneas were dissected with a 2-3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers. Beginning with the posterior chambers, the chambers were filled to excess with prewarmed Eagle’s Minimum Essential Medium (EMEM), while preventing bubble formation. The corneal holder was equilibrated at 32±1°C for at least one hour.

QUALITY CHECK OF THE ISOLATED CORNEAS: After the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Corneas exhibiting macroscopic tissue damage (e.g. scratches, pigmentation, neovascularisation) or an opacity >7 opacity units were discarded. The mean opacity of all equilibrated corneas was calculated by use of an opacitometer. A minimum of three corneaswith opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment, solvent and positive control groups (three corneas/group).

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: 0.9% sodium chloride solution

POSITIVE CONTROL USED: 1% NaOH solution in aqua ad iniectabilia

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes: 2 hours

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the exposure period the epithelium was washed with EMEM containing phenol red at least three times. Washing was repeated until no test item or discolouration (yellow or purple) of phenol red was visible. No discolouration of phenol red was visible after exposure of the test item. The corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber. The chamber was then filled with EMEM without phenol red.
- POST-EXPOSURE INCUBATION: After rinsing, the corneas were incubated at 32±1°C for two hours. After this post-exposure incubation period, the corneas were examined.

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 resulting in opacity values measured on a continuous scale.
- Corneal permeability: 1 mL sodium fluorescein solution (5 mg/mL in 0.9% sodium chloride solution) was added to the anterior chamber (epithelial surface) while the posterior chamber (endothelial surface) was refilled with fresh EMEM. The holder was incubated in a horizontal position at 32±1°C for 90±5 minutes. The amount of sodium fluorescein that crossed from the anterior to the posterior chamber was measured quantitatively using a microplate reader. Measurements at 490 nm were recorded as optical density (OD490). The fluorescein permeability values were determined using OD490 values based upon a visible light spectrophotometer using a standard 1 cm path length.
- Others (e.g, pertinent visual observations, histopathology): Corneal injury was assessed by evaluating the opacity and permeability of the cornea.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: As specified in the guideline
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
-0.492
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:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: see Table 1

The corneas treated with the negative control item 0.9% sodium chloride solution revealed a mean opacity value of 0.359 ± 0.638 and a mean permeability value of 0.015 ± 0.009. The calculated IVIS value of 0.579 ± 0.521 was well below the cut-off value of 3 (UN GHS no category).

The corneas treated with the positive control item 1% NaOH in aqua ad iniectabilia revealed a mean opacity value of 72.589 ± 21.979 and a mean permeability value of 1.654 ± 0.124 compared to the solvent control. The calculated IVIS value of 97.399 ± 21.031 was within two standard deviations of the current historical mean and well above the cut-off value of 55. Hence, the acceptance criteria for the test were fulfilled.

Following treatment with “Karstedt concentrate” a mean opacity of -0.412 ± 0.023 and a mean permeability value of -0.005 ± 0.001 compared to the negative control were determined. The calculated IVIS of -0.492 ± 0.005 is below the cut-off value of 3 (UN GHS no category). Hence, the test item did not show severely irritant or corrosive properties and consequently it is not classified as a severe irritant and is not corrosive according to UN GHS classification.

The IVIS values are given in the table below:

         Cornea No.

 Opacity (opacity units)

 Permeability (OD)

 IVIS      

 Per Cornea   

 Per group   

 Mean

 SD

 0.9% NaCl      

 1  -0.279  0.025  0.096  0.579        0.521      
 2  0.359  0.010  0.509
 3  0.996  0.009  1.131
 1% NaOH        4  96.215  1.626  120.605  97.399        21.031      
 5  68.804  1.546  91.994
 6  52.749  1.790  79.599
 Karstedt concentrate        7  -0.438  -0.004  -0.498  -0.492        0.005      
 8  -0.399  -0.006  -0.489
 9  -0.399  -0.006  -0.489

SD: standard deviation

OD: optical density

Interpretation of results:
GHS criteria not met
Conclusions:
In an in vitro bovine corneal opacity and permeability assay conducted in accordance with OECD Test Guideline 437, and to GLP, "Karstedt concentrate" did not induce a response indicative of a corrosive or severe irritant. The calculated IVIS of -0.492 is below the cut-off value of 3 (UN GHS no category).
Executive summary:

In an in vitro bovine corneal opacity and permeability (BCOP) assay conducted in accordance with OECD Test Guideline 437 and to GLP, “Karstedt concentrate” was applied to isolated bovine corneas for 10 minutes, followed by an incubation period of 2 hours. The In Vitro Irritancy Score (IVIS) calculated from individual scores for induced opacity (decreased light transmission through the cornea) and permeability (passage of sodium fluorescein dye through the cornea) was -0.492 ± 0.005. The IVIS value is less than the cut-off value of 3 (no category) and consequently the test material is not classified as a severe irritant. The positive and negative controls were considered valid.

Under the conditions of this assay, “Karstedt concentrate” would not be classified as irritating nor corrosive to the eye according to UN GHS classification.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

No relevant human irritation/corrosion data were identified.

“Karstedt concentrate” was tested for skin irritation potential in an in vitro EpiDerm assay, using reconstructed human epidermis, conducted in accordance with OECD Test Guideline 439 and to GLP. The mean cell viability following exposure to the test substance was calculated to be greater than 50% (90.6% of the negative controls), and it was therefore considered to be non-cytotoxic and predicted to be non-irritant to skin. The positive and negative controls were considered valid (Spruth, 2016a).

 

In an in vitro bovine corneal opacity and permeability (BCOP) assay conducted in accordance with OECD Test Guideline 437 and to GLP, “Karstedt concentrate” was applied to isolated bovine corneas for 10 minutes, followed by an incubation period of 2 hours. The In Vitro Irritancy Score (IVIS) calculated from individual scores for induced opacity (decreased light transmission through the cornea) and permeability (passage of sodium fluorescein dye through the cornea) was -0.492 ± 0.005. The IVIS value is less than the cut-off value of 3 (no category) and consequently the test material is not classified as a severe eye irritant. The positive and negative controls were considered valid (Spruth, 2016b).

 

No respiratory tract irritation data were identified. A new study was not conducted as it is not a REACH Standard Information Requirement.

Justification for classification or non-classification

Based on the results of the available and reliable in vitro skin and eye irritation studies, ‘Karstedt concentrate’ does not warrant classification for skin or eye irritation, according to EU CLP criteria (EC 1272/2008).