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Skin irritation / corrosion

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Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May-August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Name: RCX 15-116
Batch No.: 15047AE2
Purity: > 99%
CAS No.: 15305-07-4
Chemical Name: Aluminium-N-nitrosophenylhydroxylamine (NPAL)
Aggregate State at RT: solid
Colour: whitish
Storage Conditions: room temperature, protected from light
Stability: instable after repeated contact to air and / or light, undergoes hydrolysis
Expiry Date: 30 Nov 2016
Safety Precautions: the routine hygienic procedures were sufficient to assure personnel health and safety.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Justification for test system used:
The EpiDerm Skin Model is a well established organotypic, three-dimensional model of the human epidermis and is used for in vitro experiments since many years. It is known for its similarity to human skin.
Vehicle:
water
Details on test system:
The test was carried out with the reconstituted three-dimensional human skin model EpiDerm™ (MatTek). This skin model consists of normal (non-cancerous), human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts (Millicell™). The EpiDerm™ skin model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous, granular and cornified layers analogous to those found in vivo.

Upon receipt of the EpiDerm - kit, the tissues were transferred into 6-well plates containing 900 µL prewarmed assay medium per well. The 6-well plates were pre-incubated in a humidified incubator at 37 ± 1 °C, 5.0% CO2 / 95% air for at least 1 h. Then the medium was replaced by 900 µL fresh assay medium. The 6-well plate used for the 3 min experiment was placed back into the incubator. The other plate was used for the 60 min treatment. About 1 h before the end of the first treatment period the MTT solution was prepared by mixing the MTT concentrate with the MTT diluent and pre-warmed in the incubator.

60 min experiment: the tissues were treated with each dose group in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. Then the 6-well plate was incubated at 37 ± 1 °C, 5.0% CO2 / 95% air.
3 min experiment: the tissues were treated with each dose group in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. A constant time interval of 20 sec. was kept between dosing.

After 3 min of application, with forceps, the first insert was removed from the 6-well plate. with forceps Using a wash bottle the tissue was gently rinsed about 20 times with PBS (phospate buffered saline) to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. The insert was placed in a prepared 24-well “holding plate“ containing 300 µL pre-warmed assay medium per well. All inserts were treated in the same manner. Then the inserts were transferred into a prepared 24-well “MTT assay plate“ containing“containing 300 µL prewarmed MTT solution. The plate was incubated for 3 h at 37 ± 1 °C, 5.0% CO2 / 95% air. 3 min and 60 min experiment: after the 3 h MTT incubation period the MTT solution was aspirated. The wells were refilled with PBS and the PBS was aspirated. The rinsing was repeated twice and the tissues were dried. Then the inserts were transferred into new 24-well “extraction plates“. 2 mL of isopropanol were pipetted into each insert, thus the insert was covered from both sides. The extraction plates were sealed in zip-bags to inhibit isopropanol evaporation. Extraction was carried out over night without shaking at room temperature. After the extraction period the inserts were pierced with an injection needle to allow the extracts to run through the tissues into the corresponding wells. Then the inserts were discarded and the extraction plates were placed on a shaker for 15 min. Per each tissue 3 x 200 µL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm without reference wavelength in a plate spectrophotometer.
Amount/concentration applied:
25 mg of the test item was applied directly atop the EpiDerm™ tissue using an application spoon avoiding compression of the test item. To ensure good contact with the skin the test item was moistened with 25 µL H2O. The volume of H2O was increased and the test item was spread to match size of the tissue.
Duration of treatment / exposure:
The test was performed on a total of 4 tissues per dose group, 2 replicates for each treatment period (3 min and 60 min exposure time).
Number of replicates:
2 replicates for each treatment period
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min
Value:
>= 50
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min
Value:
>= 15
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
GHS criteria not met
Conclusions:
The test item did not show corrosive effects in the.EpiDerm™ model, a reconstituted three-dimensional human epidermis model.
Executive summary:

In the present study, the skin corrosivity potential of RCX 15 -116 was analysed. Since corrosive chemicals are cytotoxic to the stratum corneum of the epidermis the cytotoxic effects of the test item on EpiDerm™, a reconstituted three-dimensional human epidermis model, were determined. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 3 and 60 min exposure period and compared to those of the concurrent negative controls.

The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was³ 50% (97.2%) after 3 min treatment and³ 15% (104.9%) after 60 min treatment. The controls confirmed the validity of the study. The mean OD570of the two negative control tissues was 0.8 for each exposure period. The mean relative tissue viability (% negative control) of the positive control was < 15% (6.5%) after 60 min treatment. In the range of 20 – 100% viability, the coefficient of variation (CV) of replicate tissues of all dose groups was£ 30% (0.2% - 12.9%).

In conclusion, under the given conditions, the test item showed no corrosive effects. The test item is therefore classified as "non-corrosive".

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June-August 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)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Name: RCX 15-116
Batch No.: 15047AE2
Purity: > 99%
CAS No.: 15305-07-4
Chemical Name: Aluminium-N-nitrosophenylhydroxylamine (NPAL)
Aggregate State at RT: solid
Colour: whitish
Storage Conditions: room temperature, protected from light
Stability: instable after repeated contact to air and / or light, undergoes hydrolysis
Expiry Date: 30 Nov 2016
Safety Precautions: the routine hygienic procedures were sufficient to assure personnel health and safety.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
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 closely mimics the biochemical and physiological properties of the upper parts of the human, i.e. the epidermis.
Vehicle:
other: Dulbecco’s phosphate buffered saline (DPBS)
Details on test system:
The test was carried out with the reconstituted three-dimensional human skin model EpiDerm™ (MatTek). This skin model consists of normal human epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts (Millicell™). The EpiDerm™ epidermis model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous and granular layers and a multi-layered stratum corneum analogous to patterns found in vivo.

Experimental Procedure
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. For quantitative correction of results, two killed tissues were treated with 25 mg of the test item (KT) and two killed tissues were left untreated as a control (KU), respectively. All steps were performed exactly as described in the chapter below.
NSMTT was then calculated relative to the negative control of living tissues (NK) according to the following formula:
NSMTT [%] = [(ODKT - ODKU)/ODNK] * 100
If the test item is classified as non-irritant and if non-specific MTT reduction is ≤ 30% relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) of the test item treated living tissues TM was corrected according to the following formula:
TODTT = ODTM – (ODKT – ODKU)

If non-specific MTT reduction is > 30% relative to the negative control of living epidermis, the test item is considered as incompatible with the test method.
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. For quantitative correction of results, the test was performed using two additional living tissues treated with 25 mg of the test item (TVT) and one living tissue treated with 30 µL of the negative control (DPBS, UVT), respectively, in parallel to the main experiment. All steps were performed exactly as described in the chapter below, except for the MTT-staining of the test item treated with tissues, which were incubated in medium without MTT. The non-specific colour of additional viable tissues (NSCliving) was then calculated according to the following formula:
NSCliving [%] = [ODTVT/ODUVT]*100

If NSCliving is ≤ 5% relative to the negative control of living epidermis, no correction of the results is necessary. If NSCliving is > 5% and ≤ 30% relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) was corrected according to the following formula:
TODTT = ODTM – ODTVT

If NSCliving is > 30% relative to the negative control of living epidermis, the test item is considered as incompatible with the test method.

For test items which are classified as non-irritant and which act as non-specific MTT-reducers and show non-specific colouring of living tissues, a third control for non-specific colour in killed tissues (NSCkilled) was performed to avoid a possible double-correction for colour interference. Therefore, two killed tissues were treated with 25 mg of the test item (TKT) and one killed tissue was treated with the negative control (DPBS; UKT), respectively. All steps were then performed exactly as described in the chapter below, except for the MTT-staining of the test item treated with tissues, which was incubated in medium without MTT. The non-specific colour of additional viable tissues (NSCkilled) was then calculated according to the following formula:
NSCkilled [%] = [ODTKT/ODUKT]*100

The true tissue viability was then calculated as the percent tissue viability obtained with living tissues minus NSMTT minus NSCliving plus NSCkilled. If the coloured test material or MTT reducing substance is classified as “irritant” i.e. mean tissue viability is < 50%, the correction procedures are not necessary.
Upon receipt of the EpiDermTM, the tissues were inspected visually and transferred into 6-well plates containing 0.9 mL assay medium per well. The surface was dried using a sterile cotton tip and the plates were incubated in a humidified incubator at 37 ± 1 °C, 5.0% CO2 for 60 ± 5 min. Subsequently the tissues were transferred into new wells containing 0.9 mL pre-warmed assay medium per well and were incubated for 18 ± 3 h in a humidified incubator at 37 ± 1 °C, 5.0% CO2. After this pre-incubation the tissues were treated with each dose group in triplicate, starting with the negative control. Start time was recorded with dosing of the first tissue staggered in e.g. one-minute intervals. After dosing of all tissues, all plates were transferred to the incubator for 35 ± 1 min. Then all plates were removed from the incubator and placed under the sterile flow until the 60 ± 1 min incubation time of the first dosed tissue was over. Then the tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface, staggered again in e.g. one-minute intervals. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. Excess DPBS was removed by blotting the bottom with blotting paper. The inserts were placed in prepared new 6-well plates containing 0.9 mL pre-warmed fresh assay medium per well and the tissue surface was dried using a sterile cotton tip. The 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 0.9 mL fresh assay medium and incubated for additional 18 ± 2 h. After this post-incubation period the bottom of the inserts were blotted on sterile blotting paper and the inserts were transferred in a prepared 24-well plate containing 300 µL pre-warmed MTT medium. This plate was incubated for 3 h  5 min at 37 ± 1 °C, 5.0% CO2, humidified to 95%. After the MTT incubation period, the tissues were rinsed three times with DPBS and afterwards placed on blotting paper to dry. The tissues were transferred into a new 24-well plate 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 h with gentle shaking on a plate shaker. Before using the extracts, the plate was shaken for at least 15 min 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. OD was measured at 570 nm with a filter band pass of maximum ± 30 nm without reference wavelength in a plate spectrophotometer using isopropanol as a blank.
Control samples:
yes, concurrent vehicle
yes, concurrent positive control
Amount/concentration applied:
Firstly, 25 µL of sterile DPBS was applied to the epidermal surface in order to improve the contact between the powder and the epidermis. Afterwards, 25 mg (39 mg/cm2) of the test item was applied directly atop the EpiDerm™ 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.The test was performed on a total of 3 tissues per dose group.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min
Value:
> 50
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
42 h
Value:
> 50
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Interpretation of results:
GHS criteria not met
Conclusions:
The test item did not showed irritant effects in the EpiDerm™-Standard Model (EPI-200™), a reconstituted three-dimensional human epidermis model.
Executive summary:

In the present study the skin irritant potential of RCX 15-116 was analysed. The EpiDerm™-Standard Model (EPI-200™), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404, to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 60 min exposure and 42 h post-incubation periodand compared to those of the concurrent negative controls.

In this study under the given conditions the test item showed no irritant effects. The relative mean tissue viability after 60 min of exposure and 42 h post-incubation was> 50%. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

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
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)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Name: RCX 15-116
Batch No.: 15047AE2
Purity: > 99%
CAS No.: 15305-07-4
Chemical Name: Aluminium-N-nitrosophenylhydroxylamine (NPAL)
Aggregate State at RT: solid
Colour: whitish
Storage Conditions: room temperature, protected from light
Stability: instable after repeated contact to air and / or light, undergoes hydrolysis
Expiry Date: 30 Nov 2016
Safety Precautions: the routine hygienic procedures were sufficient to assure personnel health and safety.
Species:
other: cows
Vehicle:
physiological saline
Amount / concentration applied:
750 µL of the pure test item preparation or the control substance was applied.
Duration of treatment / exposure:
fter 4 hours ± 5 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an illuminance measurement was performed. Also, each cornea was observed visually, no pertinent observations were recorded. After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).
Number of animals or in vitro replicates:
3 corneas for the test item, 3 corneas as negative controls treated with physiological saline 0.9% NaCl, 3 corneas as positive controls treated with imidazole 20% in physiological saline 0.9% NaCl.

The BCOP assay is considered to be valid if the in vitro irritation score obtained with the positive control falls within the two standard deviations of the current historical mean. The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.
Details on study design:
Preparation of the Corneas
The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany. On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated. The eyes were carefully examined for defects and any defective eyes were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C.

Calibration of the Opacitometer
The opacitometer (BASF-OP3.0, Duratec GmbH) was switched on at least 15 min before starting the calibration procedure. The filter holder was placed into the opacitometer and the readout was adjusted to 1000 lux ± 10 lux using the “Calibrate”-turning knob. For calibration the glass filter F2 was introduced into the filter holder. The readout lied in the range between 540-560 lux. To test the linearity of the measurement, two additional calibration filters, glass filter F3 and glass filter F4, were measured. For these glass filters, the opacitometer displayed values between 300-310 lux and between 95-105 lux. The calibration procedure was performed before the test and is documented in the raw data.

Treatment of the Corneas
After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI. An initial measurement was performed on each of the corneas using the opacitometer. Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay. The medium was removed from the anterior chamber and replaced with the test item or control. 750 µL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method). After 4 hours ± 5 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an illuminance measurement was performed. Also, each cornea was observed visually, no pertinent observations were recorded. After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).
Irritation parameter:
cornea opacity score
Value:
0.63
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Value:
1.02
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Irritation parameter:
other: permeability
Value:
0.026
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Interpretation of results:
GHS criteria not met
Conclusions:
RCX 15-116 did not show any irritancy potential in the bovine corneal opacity and permeability assay.
Executive summary:

The eye irritancy potential of RCX 15-116 was investigated in the bovine corneal opacity and permeability assay. Technically not possible to gain a 20% concentration, therefore the test item was applied directly.

The following mean in vitro irritation score was calculated: 1.02. Therefore the test item does not need to be classified as an eye irritant.

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid. The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

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