Registration Dossier

Administrative data

Description of key information

Skin irritation and corrosion - not irritating or corrosive

Eye irritation - not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 January 2015 - 16 January 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This method is approved by international regulatory agencies as a replacement for the identification of corrosives in the in vivo Rabbit skin assay (OECD No. 404) and is specifically approved (OECD 431) as a replacement for the in vivo skin corrosivity test.
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
Due to technical reason (upgrade of the plate reader), plate was measured at 540 and 570 nm, instead of 540 nm as indicated in the Study Plan. As the maximum of the absorption peak is at 570 nm, those results are reported, however all the data will be kept and archived in the raw data binder. The measurements on the two different wavelength resulted identical conclusion.

Due to unscheduled delay of reporting, the Draft Report was issued two days later than indicated in the Study Plan. However, this fact had no impact on the results or integrity of the study.
Deviations:
yes
Remarks:
This fact was considered not to adversely affect the results or integrity of the study.
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
Due to technical reason (upgrade of the plate reader), plate was measured at 540 and 570 nm, instead of 540 nm as indicated in the Study Plan. As the maximum of the absorption peak is at 570 nm, those results are reported, however all the data will be kept and archived in the raw data binder. The measurements on the two different wavelength resulted identical conclusion.

Due to unscheduled delay of reporting, the Draft Report was issued two days later than indicated in the Study Plan. However, this fact had no impact on the results or integrity of the study.
Deviations:
yes
Remarks:
This fact was considered not to adversely affect the results or integrity of the study.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch number: 141016
- Appearance: Blue powder
- Purity: ≥85.5%
- Expiry date: 19 November 2018
- Storage conditions: Controlled Room Temperature (15-25°C, below 70 RH%)
Test system:
human skin model
Cell type:
non-transformed keratinocytes
Cell source:
other: Main basal, supra basal, spinous and granular layers and a functional stratum corneum
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN-SM
- Tissue batch number(s): 15 MAIN3 002
- Expiration date: 21 Jan 2015

KILLED EPIDERMIS
- Tissue batch number(s): 14-EKIN-043
- Expiration date: 17 November 2014

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 25.1-26.4°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 mL PBS solution
- Observable damage in the tissue due to washing: N/A
- Modifications to validated SOP:

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Batch no: 10175554
- Expiration date: 02 May 2023
- Wavelength:

NUMBER OF REPLICATE TISSUES:
In this assay, two replicates per test item per time point were used. Two negative controls and two positive controls were also run in the assay. As the test item was coloured, two additional test item-treated tissues were used for the non specific OD evaluation. Furthermore, as the test item had an MTT interacting potential, two additional test item-treated killed epidermis and two negative control treated killed epidermis were used in the study.
Control samples:
yes, concurrent negative control
yes, concurrent no treatment
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20mg
- applied as is, but 100 μL physiological saline was added to the test item to ensure good contact with the epidermis.

NEGATIVE CONTROL
Physiological saline
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): 0.9% (w/v) NaCl solution
- Batch no: 19GI12GF
- Expiry Date: 31 Aug 2018


POSITIVE CONTROL
Glacial acetic acid
- Amount(s) applied (volume or weight): 50 μL
- Batch no: 12D120031
- Expiry date: 30 Apr 2015
Duration of treatment / exposure:
4h at room temperature
Duration of post-treatment incubation (if applicable):
3h
Number of replicates:
2 per control group (plus 2 positive and negative control on killed epidermis)
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
94.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Interpretation of results:
GHS criteria not met
Conclusions:
Following exposure with test item, the mean cell viability was 94.7% compared to the negative control (after adjustment for non-specific MTT reduction).
This is above the threshold of 35%, therefore the test item was considered as being non-corrosive.

The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN model test with test item, the results indicate that the test item is not corrosive to the skin.
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 March 2015 - 27 March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
replacement for the identification of irritants / corrosives in the in vivo Rabbit skin assay (OECD No. 404).
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Due to technical reason (upgrade of the plate reader), plate was measured at 540 and
570 nm, instead of 540 nm as indicated in the Study Plan. As the maximum of the
absorption peak is at 570 nm, those results are reported, however all the data will be
kept and archived in the raw data binder. The measurements on the two different
wavelength resulted identical conclusion (mean relative viability values differed less
than 0.5%); thus, this fact was considered not to adversely affect the results or
integrity of the study.
Due to the interfering colour of the test item (dark blue) the plate was also measured
at 690 nm, in order to more precisely evaluate the possible amount of test item
remained on the surface on the skin units and the resulted non-specific colour.
Deviations:
yes
Remarks:
This fact was considered not to adversely affect the results or integrity of the study.
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Due to technical reason (upgrade of the plate reader), plate was measured at 540 and
570 nm, instead of 540 nm as indicated in the Study Plan. As the maximum of the
absorption peak is at 570 nm, those results are reported, however all the data will be
kept and archived in the raw data binder. The measurements on the two different
wavelength resulted identical conclusion (mean relative viability values differed less
than 0.5%); thus, this fact was considered not to adversely affect the results or
integrity of the study.
Due to the interfering colour of the test item (dark blue) the plate was also measured
at 690 nm, in order to more precisely evaluate the possible amount of test item
remained on the surface on the skin units and the resulted non-specific colour.
Deviations:
yes
Remarks:
This fact was considered not to adversely affect the results or integrity of the study.
Qualifier:
according to
Guideline:
other: EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals
Version / remarks:
Due to technical reason (upgrade of the plate reader), plate was measured at 540 and
570 nm, instead of 540 nm as indicated in the Study Plan. As the maximum of the
absorption peak is at 570 nm, those results are reported, however all the data will be
kept and archived in the raw data binder. The measurements on the two different
wavelength resulted identical conclusion (mean relative viability values differed less
than 0.5%); thus, this fact was considered not to adversely affect the results or
integrity of the study.
Due to the interfering colour of the test item (dark blue) the plate was also measured
at 690 nm, in order to more precisely evaluate the possible amount of test item
remained on the surface on the skin units and the resulted non-specific colour.
Deviations:
yes
Remarks:
This fact was considered not to adversely affect the results or integrity of the study.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
Batch number: 141016
Appearance: Blue powder
Purity: ≥85.5%
Expiry date: 19 November 2018
Storage conditions: Controlled Room Temperature (15-25°C, below 70 RH%)




Test system:
human skin model
Cell type:
non-transformed keratinocytes
Cell source:
other: Main basal, supra basal, spinous and granular layers and a functional stratum corneum
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN-SM
- Tissue batch number(s): 15-EKIN-012
- Expiry date: 30 Mar 2015
- Date of initiation of testing: 25 Mar 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- MTT volume 0.2mL /per well
- Incubation time: 3h
- Wavelength:570±30 nm and 690 nm

NUMBER OF REPLICATE TISSUES:
three units per control group

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
The viability expressed as a % relative to the negative control.
If the mean relative viability after 15 minutes exposure and 42 hours post incubation ≤ to 50% of the negative control, the test item is considered to be irritant to skin.
Control samples:
yes, concurrent negative control
yes, concurrent no treatment
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20mg

NEGATIVE CONTROL
Phosphate Buffered Saline
- Amount(s) applied (volume or weight): 50µL

POSITIVE CONTROL
Sodium Dodecyl Sulphate Solution
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution): 5% (w/v)
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42h at 37°C
Number of replicates:
3 per control group
Duration of treatment / exposure:
15 min
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean value
Value:
100.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Interpretation of results:
GHS criteria not met
Conclusions:
Following exposure with test item, the mean relative viability was 100.1% compared to the negative control value (after adjustment for non-specific MTT reduction). This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN model test with test item, the results indicate that the test item is non-irritant to skin.
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 May 2015 - 08 May 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2500 (Acute Dermal Irritation)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch number: 141016
- Appearance: Blue powder
- Purity: ≥85.5%

- Storage conditions: Controlled Room Temperature (15-25°C, below 70 RH%)
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 14 – 15 weeks old
- Weight at study initiation: 3538 – 3754 g
- Housing: individually housed
- Diet ad libitum
- Water ad libitum
- Acclimation period: at least 34 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 – 22.4 °C
- Humidity (%): 28 – 69 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Type of coverage:
semiocclusive
Preparation of test site:
shaved
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g

Duration of treatment / exposure:
4 hours
Observation period:
Animals were examined for signs of erythema and oedema, and the responses scored at 60 minutes and then at 24, 48 and 72 hours after patch removal.
Number of animals:
3 males
Details on study design:
TEST SITE
- Area of exposure: 6 cm² area of intact skin

- Type of wrap if used: plastic

REMOVAL OF TEST SUBSTANCE
- Washing (if done): removed with water at body temperature
- Time after start of exposure: 4 hours

OBSERVATION TIME POINTS
(indicate if minutes, hours or days) Animals were examined for signs of erythema and oedema, and the responses scored at 60 minutes and then at 24, 48 and 72 hours after patch removal

SCORING SYSTEM:
- Method of calculation: The dermal irritation scores were evaluated according to the scoring system by Draize (1959)
Irritation parameter:
erythema score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
8
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Interpretation of results:
GHS criteria not met
Conclusions:
According to the UN Globally Harmonised System of Classification and Labelling of Chemicals, B331 does not require classification as a skin irritant.
According to the classification system based on the scheme devised by Draize (1959), B331 is a "non- irritant".
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:
16 January 2015 - 16 January 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The test is described in OECD 438 and is approved by international regulatory agencies as a replacement for the identification of nonirritant, corrosives/severe irritants in the in vivo Rabbit Eye Assay (OECD 405).
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye 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.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch number: 141016
- Appearance: Blue powder
- Purity: ≥85.5%
- Expiry date: 19 Nov 2018
- Storage conditions: Controlled Room Temperature (15-25oC, below 70 RH%)
Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni út. 129., Hungary)

- Number of animals: 7 treatements (3 test item, 3 positive and 1 negative control)

- Characteristics of donor animals: age - : approx. 7 weeks old

- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): heads transported to the lab at ambient temperature at the earliest convenience. The heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed. The heads were received at the lab. and processed within approximately 2 hours of collection.

- Time interval prior to initiating testing: acclimatization for approximately 45 to 60 minutes

- indication of any existing defects or lesions in ocular tissue samples: If the cornea was in good condition, the eyeball was carefully removed from the orbit for testing. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced.

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): 30mg
Duration of treatment / exposure:
10s
Observation period (in vivo):
Test eyes were evaluated at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse
Number of animals or in vitro replicates:
7 (3 test item treated, 3 positive control and 1 negative control)
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES

NUMBER OF REPLICATES

NEGATIVE CONTROL USED
30 μL of physiological saline (Salsol solution, 0.9% (w/v) NaCl solution)

POSITIVE CONTROL USED
30 mg Imidazole

APPLICATION DOSE AND EXPOSURE TIME
10s

OBSERVATION PERIOD
evaluated at approximately 30, 75, 120, 180 and 240 minutes

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 mL physiological saline at ambient temperature
- Indicate any deviation from test procedure in the Guideline

METHODS FOR MEASURED ENDPOINTS:
Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at base line (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.
Irritation parameter:
percent corneal swelling
Run / experiment:
mean at up to 75 min
Value:
14.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
mean at up to 240min
Value:
27.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
mean
Value:
2.33
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Run / experiment:
mean
Value:
2.33
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
other:
Conclusions:
Based on this in vitro eye irritation in the isolated chicken eyes test with test item, the test item is not classified as a severe irritant and not classified as nonirritant. It is concluded that an in vivo study is required for proper classification.
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 February 2015 - 04 March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch number: 141016
- Appearance: Blue powder
- Purity: ≥85.5%

- Storage conditions: Controlled Room Temperature (15-25oC, below 70 RH%)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Age at study initiation: 12 -13 weeks old (young adult)
- Weight at study initiation: 3242 g – 3529 g
- Housing: individually housed
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4 – 21.6 °C
- Humidity (%): < 15 – 43 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Vehicle:
water
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 g


Duration of treatment / exposure:
24 hours
Observation period (in vivo):
The eyes were examined at 1, 24, 48, 72 hours after treatment.
Details on study design:
REMOVAL OF TEST SUBSTANCE
As the solid test item remained in the eye sac in all animals at the one and 24 hour observation time point, the treated eye of test animals was rinsed with physiological saline solution.

SCORING SYSTEM: The eye irritation scores were evaluated according to the scoring system by Draize (1977) and OECD 405 (02 October 2012)
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
24/48/72 h
Score:
>= 0.33 - <= 0.67
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritant / corrosive response data:
Conjunctival redness, chemosis, discharge and corneal opacity were seen in each animal 1 hour after application of the test material. In each animal some conjunctival redness and discharge remained at the 24 hour observation. Slight conjunctival redness remained in the eye of one animal at the 48 hour observation but all clinical signs and signs of eye irritation had fully reversed at the 72 hour observation; in the other two animals all signs had reversed by the 48 hour observation.
Other effects:
The test item caused a local blue discoloration of the hair around the treated eye
Interpretation of results:
GHS criteria not met
Conclusions:
The test item B331, applied to rabbit eye mucosa, caused conjunctival effects at one hour after application which were fully reversible within 72 hours.
According to Regulation (EC) No 1272/2008, B331 does not require classification as an eye irritant.
According to the UN Globally Harmonised System of Classification and Labelling of Chemicals, B331 does not require classification as an eye irritant.
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

B331 was determined to be non-corrosive and non-irritating to skin in relevant in-vitro and in-vivo studies. B331 was found to be non-irritating to the eyes in a relevant in-vivo eye irritation test.

In accordance the Regulation (EC) 1272/2008 it is concluded that B331 does not meet the criteria for classification as a skin or eye irritant.