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Administrative data

Description of key information

Skin corrosion: In view of the absence of skin and eye irritation the substance is not corrosive

Skin irritation: in vitro test (OECD 439): not irritating

Eye irritation: in vitro test (OECD 438): not irritating

Respiratory irritation: Not a respiratory irritant (based on absence of human data indicating such and absence of skin and eye irritation)

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:
2015-09-02 till 2015-09-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. certificate)
Remarks:
TNO Triskelion, Utrechtseweg 48, 3700 AV, Zeist
Test system:
human skin model
Remarks:
EpiDerm™ (EPI-200) skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Source strain:
other: Not relevant
Details on test system:
The EpiDerm™ (EPI-200) skin model consisted of normal human epidermal keratinocytes from one single donor, derived from neonatal-foreskin tissue. The keratinocytes were plated on chemically modified, collagen-coated, 9 mm ID cell culture inserts (surface area 0.64 cm2). The skin models are commercially available and were obtained from MatTek In Vitro Life Science Laboratories (IVLSL), Slovakia.

Preliminary tests:
- Some chemicals are known to change into a coloured substance in aqueous conditions and consequently stain tissues during the exposure. To determine this possibility, 30 μL of the test substance was incubated in 300 μL MilliQ water for 60 min at ca. 37 ºC and 5% CO2 and a colour change was assessed visually. Some chemicals are known to non-specifically reduce MTT, resulting in a blue/purple precipitate and/or blue/purple staining of a MTT solution. To test the MTT reducing capacity of the test substance, 30 μL of the test substance was incubated in 1 mL of a MTT solution (1 mg/mL) for 185 min at ca. 37ºC and 5% CO2 and the formation of a blue/purple formazan product was assessed visually. Based on the results, the negative control and test substance were applied to frozen controls (n=2 per test group) in the in vitro skin irritation test. To test if the test substance had the potential to damage a nylon mesh, a mesh compatibility test was performed in which 30 μL of the test substance was applied to a nylon mesh. After 60 min incubation at ambient temperature, the possible interaction with the mesh was visually checked using a microscope.

Exposure to study substances:
- The skin models were topically exposed to 30 μL of the test substance, negative or positive control, at the end of the acclimatization period. Immediately after application a nylon mesh was placed on the skin model surface to facilitate an equal distribution of the study substances. Exposure was initiated at ambient temperature. After dosing the last skin model, all skin models were transferred to a humidified incubator (ca. 37ºC and 5% CO2). Frozen controls were included as required. After 36 min, the plates were removed from the incubator and kept at ambient temperature until the exposure period of 60 min was completed. Subsequently, the skin models were removed from the well, washed using an excess of PBS to remove the study substances and mesh. The skin models were transferred to a clean 6-well plate containing fresh NMM (900 μL/well) and incubated in a humidified incubator (ca. 37ºC and ca. 5% CO2). Medium was refreshed after 18 h post-exposure. Following an additional 24 h incubation period (i.e. the total post-exposure period was 42 h), viability was determined using the MTT test

MTT test:
- The MTT solution of 1 mg/mL was freshly prepared by diluting MTT concentrate five times in MTT diluent. The inserts were transferred to a 24-well plate containing 300 μL of MTT solution per well. After 178 min incubation in a humidified incubator at ca. 37 ºC and 5% CO2, the skin models were rinsed three times with PBS. The formazan product was extracted from the skin model using 2 mL MTT extractant (provided with the MTT-100 assay kit). Extraction was performed at 2-10 ºC for three days. Following extraction, the optical density was measured in triplicate in 200 μL sub fractions per well in a 96-well plate using a spectrophotometer set at 570 nm. MTT extractant was used as blank. The mean optical density (OD) was calculated and expressed as the percentage viability compared to the negative control (mean tissue viability).

Interpretation of results:
- The OD of the viable skin models was corrected for non-specific MTT reduction of the test substance according to the following formula: True OD = mean OD viable skin models treated with the test substance – mean OD frozen controls, where mean OD frozencontrols = mean OD of the frozen controls treated with the test substance – the mean OD of the frozen controls treated with the negative control.
- If the non-specific reduction of MTT by the test substance was comparable to the negative control, a correction is not required. If the non-specific reduction of MTT by the test substance was > 30% compared to the negative control, the test substance will be considered incompatible with the test (expert judgement).
- The in vitro skin corrosion test was considered valid if the OD of the negative control was ≥ 0.8 and ≤ 2.8, the OD of the blank was < 0.1, and skin models treated with the positive control showed mean tissue viability ≤ 20% compared to the negative control
- The test was considered invalid if the test did not meet these acceptance criteria.
- The test group was considered valid if the SD calculated from individual tissue viability percentages of the three replicates was ≤ 18%. Test substances showing tissue viabilities in a range of 30-70% may show standard deviation (SD) > 18%. If this was typical for the test substance, and consistent in a repeat experiment, the results were accepted, although the acceptance criterion was not met.
- The test group was considered invalid (inconclusive) if the SD calculated from individual tissue viability percentages of the three replicates was >18% and if the test was not repeated.
- The in vitro irritation potential of the test substance was determined from the relative mean tissue viabilities compared to the negative control tissues, using the following prediction model:

Mean tissue viability (% of negative control) Prediction
Mean tissue viability ≤ 50 % Irritant
Mean tissue viability > 50 % Non-irritant (No Category)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 uL
- Concentration: Undiluted
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Remarks:
% of negative control
Run / experiment:
mean
Value:
105
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Time point: 60 min. Max. score: 100.0. Remarks: (SD = 11)
Other effects / acceptance of results:
Preliminary tests
- At the end of the incubation period of the test substance in MilliQ, the solution did not significantly changed colour to blue/purple, indicating that there would be no colour interference in the test. Therefore, no additional controls were required in the in vitro skin irritation test.
- At the end of the incubation period of the test substance with a MTT solution, the MTT solution showed a purple top layer, indicating that the test substance had the potential to reduce MTT. Therefore, frozen controls were included in the in vitro skin irritation test.
- During the mesh compatibility test it was observed that the test substances did not damage the nylon mesh and therefore the nylon mesh was used in the in vitro skin irritation test to facilitate equal distribution of the test substance.

In vitro skin irritation test:
- The OD of the blank, the negative control (PBS) and the positive control (5% SDS) demonstrated the expected response. The SD calculated from individual tissue viability percentages of the three replicates was <18%. All acceptance criteria were met and therefore the study was considered valid.
- The OD of the frozen control membranes exposed to the test substance was comparable to the OD of the frozen control membranes exposed to the negative control. Therefore no data correction was applied.
Interpretation of results:
other: Not a skin irritant
Remarks:
in accordance with EU CLP (EC 1272/2008 and its updates)
Conclusions:
Under the test conditions (OECD 439 and GLP) the test substance is not considered to be a skin irritant.
Executive summary:

In accordance to OECD guideline 439 and GLP the test substance was examined for its in vitro skin irritation potential using EpiDerm™ reconstructed skin membranes. In the in vitro skin irritation test, the skin membranes were topically exposed to the undiluted test substance for 60 min. The test was performed in triplicate. Viability of the epidermal cells was assessed using the MTT test after 42 h of culture. Negative and positive controls were run in parallel. All acceptance criteria were met and therefore the study was considered valid. After exposure to the test substance the mean tissue viability was 105 ± 11 % compared to the concurrent negative control group. Based on the results obtained in the present study the test substance is not considered to be a skin irritant.

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:
2015-09-01 till 2015-09-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD 438 guideline study in compliance with GLP, available as unpublished report, no restrictions, fully adequate for assessment
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
GLP compliance:
yes (incl. certificate)
Remarks:
TNO Triskelion, Utrechtseweg 48, 3700 AV, Zeist
Species:
other: eyes of male or female chickens (ROSS, spring chickens)
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Slaughterhouse v.d. Bor, Nijkerkerveen, The Netherlands
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: approximately 1.5 - 2.5 kg
- Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.
- The preparation and validation of the eyes prior to the ICE-test were all according to OECD guideline 438.
Vehicle:
unchanged (no vehicle)
Controls:
other: Positive controls: Benzalkonium Chloride. Negative control: Phosphate buffered saline (PBS).
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 30 µL
Duration of treatment / exposure:
10 seconds
Observation period (in vivo):
0, 30, 75, 120, 180, and 240 minutes
Number of animals or in vitro replicates:
3 eyes
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: The eyes were rinsed with 20 mL saline
- Time after start of exposure: 10 seconds

SCORING SYSTEM: According to OECD 438 guideline.

TOOL USED TO ASSESS SCORE: All examinations were carried out with the hand-slit lamp microscope. Fluorescein retention was only scored at approximately 30 minutes after treatment. After the final examination, the test substance treated eyes, the negative and positive control eyes were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The corneas were embedded in paraffin wax, sectioned at ca 4 μm and stained with PAS (Periodic Acid-Schiff). The microscopic slides were subjected to histopathological examination.

CONTROLS: A negative control (30 µL physiological saline) and 3 positive controls (30 µL Benzalkonium Chloride 5%) were included.
Irritation parameter:
percent corneal swelling
Run / experiment:
mean 240 min
Value:
4
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Maximum value during the test
Irritation parameter:
cornea opacity score
Run / experiment:
mean 180 min
Value:
0.7
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Maximum value during the test
Irritation parameter:
fluorescein retention score
Run / experiment:
mean 30 min
Value:
0
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Slit-lamp examination:
The test substance caused very slight corneal swelling (4%), very slight or slight opacity (mean score of 0.7) and no fluorescein retention (mean score of 0.0). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants

Microscopic examination:
Microscopic examination of the corneas treated with the test substance revealed very slight erosion (two corneas) and slight necrosis (one cornea) of the epithelium. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities, apart from very slight vacuolation (top region) of the epithelium. The positive control BAC 5% caused moderate or severe erosion, slight vacuolation (one cornea; low region) of the epithelium, and endothelial necrosis.
Interpretation of results:
other: Not an eye irritant
Remarks:
in accordance with EU CLP (EC 1272/2008 and its updates)
Conclusions:
Under the test conditions (OECD 438 and GLP) the test substance is not considered to be an eye irritant.
Executive summary:

In accordance to OECD guideline 438 and GLP the test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) Test. In the ICE test, 3 eyes were exposed to 30 µL test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL Benzalkonium Chloride (BAC)) were tested. After the exposure the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. The test substance caused very slight corneal swelling (4%), very slight or slight opacity (mean score of 0.7) and no fluorescein retention (mean score of 0.0). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with Pinyl Iso Butyraldehyde revealed very slight erosion (two corneas) and slight necrosis (one cornea) of the epithelium. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities, apart from very slight vacuolation (top region) of the epithelium. The positive control BAC 5% caused moderate or severe erosion, slight vacuolation (one cornea; low region) of the epithelium, and endothelial necrosis. Based on these results the test substance is not considered to be an eye irritant.

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

Respiratory irritation

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

Additional information

Skin corrosion: In view of the absence of skin and eye irritation the substance is not corrosive

Skin irritation

In accordance to OECD guideline 439 and GLP the test substance was examined for its in vitro skin irritation potential using EpiDerm™ reconstructed skin membranes. In the in vitro skin irritation test, the skin membranes were topically exposed to the undiluted test substance for 60 min. The test was performed in triplicate. Viability of the epidermal cells was assessed using the MTT test after 42 h of culture. Negative and positive controls were run in parallel. All acceptance criteria were met and therefore the study was considered valid. After exposure to the test substance the mean tissue viability was 105 ± 11 % compared to the concurrent negative control group. Based on the results obtained in the present study the test substance is not considered to be a skin irritant.

 

Eye irritation

In accordance to OECD guideline 438 and GLP the test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) Test. In the ICE test, 3 eyes were exposed to 30 µL test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL Benzalkonium Chloride (BAC)) were tested. After the exposure the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. The test substance caused very slight corneal swelling (4%), very slight or slight opacity (mean score of 0.7) and no fluorescein retention (mean score of 0.0). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with Pinyl Iso Butyraldehyde revealed very slight erosion (two corneas) and slight necrosis (one cornea) of the epithelium. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities, apart from very slight vacuolation (top region) of the epithelium. The positive control BAC 5% caused moderate or severe erosion, slight vacuolation (one cornea; low region) of the epithelium, and endothelial necrosis. Based on these results the test substance is not considered to be an eye irritant.

 

Respiratory irritation

Not a respiratory irritant (based on absence of human data indicating such and absence of skin and eye irritation).

Justification for classification or non-classification

Based on the available information the test substance does not need to be classified for skin, eye and respiratory irritation in accordance with the criteria outlined in EU CLP (EC no. 1272/2008 and its amendments).