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Diss Factsheets

Administrative data

Description of key information

Based on the results from in vitro/ex-vivo/in vivo studies, the test substance is concluded to be non-irritating to the skin and eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 04, 2014 to May 18, 2014
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch No.: 5307; Purity: 99.9%; Appearance: white powder
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EPISKIN model - reconstructed human epidermis
Details on test system:
EPISKIN Reconstructed Human Epidermis Model Kit
Supplier: SkinEthic Laboratories, Lyon, France
Date received: 04 March 2014
EpiSkin Tissues (0.38 cm2) lot number: 14-EKIN-007
Maintenance Medium lot number: 14-MAIN3-009
Assay Medium lot number: 14-ESSC-008
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 mg (26.3 mg/cm2) of the test substance was applied to the epidermal surface.
Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control substance, the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). After 7-Minutes contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period. The plates were kept in the biological safety cabinet at room temperature for 15 min.
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
Triplicates for the test substance, positive and negative control
Irritation / corrosion parameter:
% tissue viability
Value:
99.7
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Direct MTT Reduction
The MTT solution containing the test substance did not turn blue which indicated that the test substance did not directly reduce MTT.

Test substance
The relative mean viability of the test substance treated tissues was 99.7% after a 15 min exposure period and 42 h post-exposure incubation period.
It was considered unnecessary to perform IL-1a analysis as the results of the MTT test were unequivocal.

Quality Criteria
The relative mean tissue viability for the positive control treated tissues was 7.5% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.8%. The positive control acceptance criterion was therefore satisfied.
The mean OD 562 for the negative control treated tissues was 0.926 and the standard deviation value of the percentage viability was 1.7%. The negative control acceptance criterion was therefore satisfied.
The standard deviation calculated from individual percentage tissue viabilities of the three identically test substance treated tissues was 5.1%. The test substance acceptance criterion was therefore satisfied.
Interpretation of results:
other: CLP criteria not met
Conclusions:
Under the study conditions, the test substance was concluded to be non-irritating to skin.
Executive summary:

An in vitro study was conducted to determine the skin irritation potential of the test substance PBBA according to OECD Guideline 439 and EU Method B.46, in compliance with GLP. Skin irritation potential was examined using the EPISKIN reconstructed human epidermis model following a treatment period of 15 min and a post-exposure incubation period of 42 h. The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test substance by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt in the substance treated tissues relative to the negative controls. To identify possible interference of the test substance with the MTT endpoint, the test substance was checked for the ability to directly reduce MTT prior to the main assay. On the day of the main test, triplicate tissues were treated with the test substance for an exposure period of 15 min. At the end of the exposure period, each tissue was rinsed before incubating for 42 h at 37°C, 5% CO2 in air. Formazan extraction was performed at Day 3. At the end of the post-exposure incubation period, each tissue was taken for MTT-loading. Thereafter, a total biopsy of each epidermis was made and placed into micro-tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. Absorbance or optical density measurements were conducted at Day 6. The optical density was measured at 562 nm. The relative mean viability of the test substance treated tissues was 99.7%, which is well above the non-irritant viability limit of >50%. Under the study conditions, the test substance is concluded to be non-irritating to skin (Warren, 2014).

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 06, 2014 to May 15, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch No.: 5307; Purity: 99.9%; Appearane: white powder
Species:
rabbit
Strain:
New Zealand White
Remarks:
Hsdlf:NZW
Details on test animals or test system and environmental conditions:
Two New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Harlan Laboratories UK Ltd., Leicestershire, UK. At the start of the study the animals weighed 2.64 or 2.83 kg and were twelve to twenty weeks old. After an acclimatization period of at least five days each animal was given a number unique within the study which was written with a black indelible marker-pen on the inner surface of the ear and on the cage label.
The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 17 to 23°C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
The rabbit is the preferred species of choice as historically used for irritation studies and is specified in the appropriate test guidelines. The number of animals used was the minimum required to achieve the objectives of the study. Testing was conducted on two animals and the response in these animals was such that exposure of a third animal would not affect classification of the test item, therefore no further testing was needed.
Type of coverage:
occlusive
Preparation of test site:
clipped
Vehicle:
water
Remarks:
0.5 mL of distilled water was added to moisten the test substance and achieve a paste
Controls:
not required
Amount / concentration applied:
0.5 g of the test substance
Duration of treatment / exposure:
Treatment duration: 3 min, 1 h and 4 h after application for the first animal, 4 h for the second animal
Observation period:
In both examinations the test sites were examined for evidence of primary irritation immediately following removal of the patches and approximately 1, 24, 48 and 72 h later.
Number of animals:
2
Details on study design:
One rabbit was initially treated. Three suitable sites were selected on the back of the rabbit. At each test site a quantity of 0.5 g of the test substance, moistened sufficiently with 0.5 mL of distilled water to achieve a paste, was introduced under a 2.5 cm x 2.5 cm cotton gauze patch. Each patch was secured in position with a strip of surgical adhesive tape. To prevent the animal interfering with the patches, the trunk of the rabbit was wrapped in an elasticated corset for the duration of the exposure period.
One patch was removed at each of three time points: 3 min, 1 h and 4 h after application. Any residual test substance was removed by gentle swabbing with cotton wool soaked in distilled water.
After consideration of the skin reactions produced in the first animal, an additional animal was treated with 0.5 g of test substance, moistened sufficiently with 0.5 mL of distilled water to achieve a paste. One patch was applied to the back of the rabbit and was allowed to remain in contact with the skin for a period of four hours. Immediately following removal of the patches and approximately 1, 24, 48 and 72 h later, the test sites were examined for evidence of primary irritation and scored according to the scale indicated in the tab for additional information. Any other skin reactions and clinical signs of toxicity, if present, were also recorded. Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.
Irritation parameter:
overall irritation score
Basis:
mean
Remarks:
2 animals
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
erythema score
Remarks:
edema score
Basis:
mean
Remarks:
2 animals
Time point:
24/48/72 h
Score:
ca. 0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
mean
Remarks:
2 animals
Time point:
24/48/72 h
Score:
ca. 0
Max. score:
4
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
3-min and 1-h semi-occluded applications of the test substance to the intact skin of one rabbit produced no evidence of skin irritation. A single 4-h, semi-occluded application of the test substance to the intact skin of two rabbits produced no evidence of skin irritation. Therefore, the primary irritation index was determined to be 0.0. No corrosive effects were noted.
Other effects:
One animal showed body weight loss and the other animal showed expected body weight gain.
Interpretation of results:
other: CLP criteria not met
Conclusions:
Under the study conditions, the test substance was concluded to be non-irritating to rabbit skin.

Executive summary:

A study was conducted to determine the skin irritation potential of the test substance PBBA, following single, 3 min, 1 and 4 h, semi-occluded applications to the intact rabbit skin, according to OECD Guideline 404 and EU Method B.4, in compliance with GLP. Three suitable sites were selected on the back of the first rabbit. At each test site, a quantity of 0.5 g of the test substance, moistened sufficiently with 0.5 mL of distilled water to achieve a paste, was introduced under a 2.5 cm x 2.5 cm cotton gauze patch. One patch was removed at each of three time points: 3 min, 1 h and 4 h after application. The second animal was treated similarly with one patch remaining in contact with skin for 4 h. In both examinations, the test sites were examined for evidence of primary irritation immediately following removal of the patches and approximately 1, 24, 48 and 72 h later. Any other skin reactions and clinical signs of toxicity, if present, were also recorded. Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period. No evidence of skin irritation was observed in any of the three rabbits at any of the observation time points. The test substance produced a primary irritation index of 0.0. Under the study conditions, the test substance was concluded to be non-irritating to rabbit skin (Pooles, 2014).

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:
From April 02, 2014 to May 26, 2014
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 Ocular Corrosives and Severe Irritants)
Deviations:
yes
Remarks:
see 'Any other information on materials and methods'
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch No.: 5307; Purity: 99.9%; Appearance: white powder
Species:
cattle
Strain:
other:
Remarks:
in vitro bovine cornea study
Details on test animals or tissues and environmental conditions:
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were refrigerated on arrival and used within 24 h of receipt.
Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
For the purpose of this study the test substance was prepared as a 20% w/v solution in 0.9% w/v sodium chloride solution. The test substance was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
Duration of treatment / exposure:
240 min
Duration of post- treatment incubation (in vitro):
First incubation, with the test substance for 240 min. Subsequently a post treatment opacity reading was taken and each cornea was visually observed.
Second incubation: 1 mL of sodium fluorescein solution (5 mg/mL) at 32 ± 1ºC for 90 min.
Number of animals or in vitro replicates:
Three corneas were allocated to the negative control. Three corneas were also allocated to the test substance and three corneas to the positive control substance.
Details on study design:
Preparation of Corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed (epithelial side uppermost) in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s minimum essential medium (MEM) and plugged. The holders were incubated at 32 ± 1ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete MEM.A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer (Appendix 1 in tab for other information). The average opacity for all corneas was calculated.
Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test substance and three corneas to the positive control substance.

Treatment of Corneas
The MEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test substance preparation or control substance were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the substance over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1ºC for 240 minutes.
At the end of the exposure period the test substance preparation and control substances were removed from the anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM. The anterior and posterior chambers were refilled with fresh complete MEM. A post treatment opacity reading was taken and each cornea was visually observed.

Application of Sodium Fluorescein
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1ºC for 90 min.

Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 μL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.

Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.
Irritation parameter:
in vitro irritation score
Run / experiment:
The two endpoints opacity and permeability were combined in an empirically derived formula to generate an in vitro irritancy score (IVIS)
Value:
3.1
Negative controls validity:
valid
Remarks:
in vitro irritation score: 2.5
Positive controls validity:
valid
Remarks:
in vitro irritation score: 83.8
Remarks on result:
other: The result of this study has identified the test substance has not causing serious eye damage, but they do not permit conclusion that the test substance does not require classification for eye irritation.

The test substance is classified according to the prediction model below:

IVIS

CLASSIFICATION

3

No category. Not requiring classification to UN GHS or EU CLP

> 3;55

No prediction of eye irritation can be made

> 55

Category 1. UN GHS or EU CLP Causes serious eye damage

Acceptance criteria:

The positive control In Vitro Irritancy Score was within the range of 73.8 – 103.6. The positive control acceptance criterion was therefore satisfied.

The negative control gave opacity of ≤4.7 and permeability ≤0.085. The negative control acceptance criteria were therefore satisfied.

Interpretation of results:
study cannot be used for classification
Conclusions:
Under the study conditions, although the test substance did not cause serious eye damage to the bovine corneas, based on the results no prediction no prediction could be made about eye irritation potential of the test substance
Executive summary:

An ex vivo study was conducted to determine the corneal damage potential of the test substance according to OECD Guideline 437, in compliance with GLP. In this in vitro method, damage is assessed by quantitative measurements of changes in corneal opacity and permeability. Bovine corneas were collected from slaughtered cattle which were between 12 and 60 months old. One valid experiment was performed with three replicates for negative control, positive control and the test substance. 0.75 mL of the test substance was applied at the concentration of 20 % w/v in 0.9 % w/v sodium chloride solute in a way that as much as possible of the corneas surface was covered. Subsequently, corneas were incubated for 240 min at 32 ± 1°C in a corneal holder. At the end of the exposure period, the test and control substances were removed from the anterior chamber and the corneas were rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM. The anterior and posterior chambers were refilled with fresh complete MEM. A post treatment opacity reading was taken and each cornea was visually observed. Following the opacity measurement, the permeability of the corneas to sodium fluorescein was evaluated. The medium was replaced with 1 mL of sodium fluorescein solution (5 mg/mL) and corneas were incubated again at 32 ± 1ºC for 90 min. After incubation 360 μL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using an Anthos 2001 microplate reader. The two endpoints opacity and permeability were combined in an empirically derived formula to generate an in vitro irritancy score (IVIS). The IVIS of the test substance was determined to be 3.1 which is well below the corrosive limit of 55 and above the non-corrosive limit of 3; therefore, no predictions could be made. Therefore, based on EU CLP criteria, no prediction could be made about eye irritation potential of the test substance. The IVIS of the positive control was within the range of 31.6 to 58.7, therefore acceptance criterion was satisfied. The negative control gave opacity of ≤3.0 and permeability ≤0.077, therefore the negative control acceptance criteria were satisfied. The quality criteria required for acceptance of results in the test were satisfied. Under the study conditions, although the test substance did not cause serious eye damage to the bovine corneas, based on the results no prediction no prediction could be made about eye irritation potential of the test substance (Warren, 2014).

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From February 06, 2014 to May 20, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch No.: 5307; Purity: 99.9%; Appearance: white powder
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Leicestershire, UK
- Age at study initiation: 12-20 weeks
- Weight at study initiation: 2.58 or 2.95 kg
- Housing: individually in suspended cages
- Diet: ad libitum, 2930C Teklad Global Rabbit diet
- Water: ad libitum
- Acclimation period: at least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23°C
- Humidity (%): 30-70%
- Air changes (per hr): 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12/12
- The animals were individually housed in suspended cages. Free access to mains drinking water and food 2930C Tekland Global rabbit diet supply was allowed throughout the study. The drinking water and diet are considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
Vehicle:
unchanged (no vehicle)
Controls:
other: the second eye of each rabbit
Amount / concentration applied:
0.1 mL (64 mg)
Duration of treatment / exposure:
72 h
Observation period (in vivo):
after 1, 24, 48 and 72 h
Number of animals or in vitro replicates:
2
Details on study design:
For the purpose of the study the test substance was used as supplied.

The pH of the test substance was determined prior to commencement of the study and found to be as follows:
pH Measurement of 10 % w/w aqueous preparation of the test substance:
- immediately: 6.29
- after 10 minutes: 6.22

Procedure
Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used. Initially, a single rabbit was treated. A subcutaneous injection of buprenorphine 0.01 mg/kg was administered 60 minutes prior to test substance application to provide a therapeutic level of systemic analgesia. Five minutes prior to test substance application, a pre-dose anesthesia of ocular anesthetic (two drops of 0.5 % tetracaine hydrochloride) was applied to each eye.

A volume of 0.1 mL of the test substance, which was found to weigh approximately 64 mg (as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctiva! sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test substance, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test substance, an assessment of the initial pain reaction was made. Eight hours after test substance application, a subcutaneous injection of post-dose analgesia, buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg, was administered to provide a continued therapeutic level of systemic analgesia. The treated animal was checked for signs of pain and suffering approximately 12 hours later. No further analgesia was required. After consideration of the ocular responses produced in the first treated animal, a second animal was similarly treated. Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation. Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope. Any clinical signs of toxicity, if present, were also recorded. Individual body weights were recorded on day 0 (the day of dosing) and at the end of the observation period.


Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
2 animals
Time point:
24/48/72 h
Score:
ca. 0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
other: cornea / area of cornea involved
Basis:
mean
Remarks:
2 animals
Time point:
24/48/72 h
Score:
ca. 0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
mean
Remarks:
2 animals
Time point:
24/48/72 h
Score:
ca. 0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
mean
Remarks:
2 animals
Time point:
24/48/72 h
Score:
ca. 0.5
Max. score:
3
Reversibility:
fully reversible within: 72 h
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
2 animals
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 72 h
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Remarks:
discharge
Basis:
mean
Remarks:
2 animals
Time point:
24/48/72 h
Score:
ca. 0.17
Max. score:
3
Reversibility:
fully reversible within: 48 h
Remarks on result:
no indication of irritation
Irritation parameter:
other: Group mean score
Remarks:
(corresponding to the highest group mean)
Basis:
mean
Remarks:
2 animals
Time point:
other: 1 h
Score:
ca. 9
Max. score:
110
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Other effects:
One animal showed body weight loss and the other animal showed expected gain in body weight during the study.

No corneal or iridial effects were noted during the study. Moderate conjunctival irritation was noted in all treated eyes one hour after the treatment. Minimal conjunctival irritation was noted in all treated eys at the 24-h observation and in one treated eye at the 48 -h observation. One treated eye appeared normal at the 48 -h observation and the other treated eye appeared normal after 72 -h observation.

Interpretation of results:
other: CLP criteria not met
Conclusions:
Under the study conditions, the test substance is concluded to be mildly irritating, but not meeting the CLP criteria for eye irriation classification.
Executive summary:

A study was conducted to determine the eye irritation potential of the test substance PBBA in New Zealand White rabbits according to OECD Guideline 405 and EU Method B.5, in compliance with GLP. A volume of 1 mL (64 mg) of the test substance was placed into the conjunctival sac of right eye of a first rabbit. The left eye remained untreated for control purposes. Immediately after administration then 12 h later, a pain and suffering assessment was made. After consideration of the ocular responses produced in the first animal, a second animal was similarly treated. An assessment of ocular damage/irritation was made approximately 1, 24, 48 and 72 h following treatment, according to a numerical scale. A single application of the test substance to the non-irrigated eye of two rabbits produced moderate conjunctival irritation. One treated eye appeared normal at the 48 h observation and the other appeared normal at the 72 h observation. Under the study conditions, the test substance produced a maximum group mean score of 9.0 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system. However, based on the individual mean scores for corneal opacity, iritis and conjunctival redness, the test substance does not warrant a classification for eye irritation according to EU CLP (EC 1272/2008) criteria (Pooles, 2014).

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin:

In vitro, human skin model

An in vitro study was conducted to determine the skin irritation potential of the test substance PBBA according to OECD Guideline 439 and EU Method B.46, in compliance with GLP. Skin irritation potential was examined using the EPISKIN reconstructed human epidermis model following a treatment period of 15 min and a post-exposure incubation period of 42 h. The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test substance by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt in the substance treated tissues relative to the negative controls. To identify possible interference of the test substance with the MTT endpoint, the test substance was checked for the ability to directly reduce MTT prior to the main assay. On the day of the main test, triplicate tissues were treated with the test substance for an exposure period of 15 min. At the end of the exposure period, each tissue was rinsed before incubating for 42 h at 37°C, 5% CO2 in air. Formazan extraction was performed at Day 3. At the end of the post-exposure incubation period, each tissue was taken for MTT-loading. Thereafter, a total biopsy of each epidermis was made and placed into micro-tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. Absorbance or optical density measurements were conducted at Day 6. The optical density was measured at 562 nm. The relative mean viability of the test substance treated tissues was 99.7%, which is well above the non-irritant viability limit of >50%. Under the study conditions, the test substance is concluded to be non-irritating to skin (Warren, 2014).

In vivo, rabbits

A study was conducted to determine the skin irritation potential of the test substance PBBA, following single, 3 min, 1 and 4 h, semi-occluded applications to the intact rabbit skin, according to OECD Guideline 404 and EU Method B.4, in compliance with GLP. Three suitable sites were selected on the back of the first rabbit. At each test site, a quantity of 0.5 g of the test substance, moistened sufficiently with 0.5 mL of distilled water to achieve a paste, was introduced under a 2.5 cm x 2.5 cm cotton gauze patch. One patch was removed at each of three time points: 3 min, 1 h and 4 h after application. The second animal was treated similarly with one patch remaining in contact with skin for 4 h. In both examinations, the test sites were examined for evidence of primary irritation immediately following removal of the patches and approximately 1, 24, 48 and 72 h later. Any other skin reactions and clinical signs of toxicity, if present, were also recorded. Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period. No evidence of skin irritation was observed in any of the three rabbits at any of the observation time points. The test substance produced a primary irritation index of 0.0. Under the study conditions, the test substance was concluded to be non-irritating to rabbit skin (Pooles, 2014).

Eye:

Ex-vivo bovine cornea model

An ex vivo study was conducted to determine the corneal damage potential of the test substance according to OECD Guideline 437, in compliance with GLP. In this in vitro method, damage is assessed by quantitative measurements of changes in corneal opacity and permeability. Bovine corneas were collected from slaughtered cattle which were between 12 and 60 months old. One valid experiment was performed with three replicates for negative control, positive control and the test substance. 0.75 mL of the test substance was applied at the concentration of 20 % w/v in 0.9 % w/v sodium chloride solute in a way that as much as possible of the corneas surface was covered. Subsequently, corneas were incubated for 240 min at 32 ± 1°C in a corneal holder. At the end of the exposure period, the test and control substances were removed from the anterior chamber and the corneas were rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM. The anterior and posterior chambers were refilled with fresh complete MEM. A post treatment opacity reading was taken and each cornea was visually observed. Following the opacity measurement, the permeability of the corneas to sodium fluorescein was evaluated. The medium was replaced with 1 mL of sodium fluorescein solution (5 mg/mL) and corneas were incubated again at 32 ± 1ºC for 90 min. After incubation 360 μL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using an Anthos 2001 microplate reader. The two endpoints opacity and permeability were combined in an empirically derived formula to generate an in vitro irritancy score (IVIS). The IVIS of the test substance was determined to be 3.1 which is well below the corrosive limit of 55 and above the non-corrosive limit of 3; therefore, no predictions could be made. Therefore, based on EU CLP criteria, no prediction could be made about eye irritation potential of the test substance. The IVIS of the positive control was within the range of 31.6 to 58.7, therefore acceptance criterion was satisfied. The negative control gave opacity of ≤3.0 and permeability ≤0.077, therefore the negative control acceptance criteria were satisfied. The quality criteria required for acceptance of results in the test were satisfied. Under the study conditions, although the test substance did not cause serious eye damage to the bovine corneas, based on the results no prediction no prediction could be made about eye irritation potential of the test substance (Warren, 2014).

In vivo, rabbits

A study was conducted to determine the eye irritation potential of the test substance PBBA in New Zealand White rabbits according to OECD Guideline 405 and EU Method B.5, in compliance with GLP. A volume of 1 mL (64 mg) of the test substance was placed into the conjunctival sac of right eye of a first rabbit. The left eye remained untreated for control purposes. Immediately after administration then 12 h later, a pain and suffering assessment was made. After consideration of the ocular responses produced in the first animal, a second animal was similarly treated. An assessment of ocular damage/irritation was made approximately 1, 24, 48 and 72 h following treatment, according to a numerical scale. A single application of the test substance to the non-irrigated eye of two rabbits produced moderate conjunctival irritation. One treated eye appeared normal at the 48 h observation and the other appeared normal at the 72 h observation. Under the study conditions, the test substance produced a maximum group mean score of 9.0 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system. However, based on the individual mean scores for corneal opacity, iritis and conjunctival redness, the test substance does not warrant a classification for eye irritation according to EU CLP (EC 1272/2008) criteria (Pooles, 2014).

Based on the results from in vitro/ex-vivo/in vivo studies, the test substance is concluded to be non-irritating to the skin and eyes.

Justification for classification or non-classification

Based on the results of in vitro and in vivo testing, the substance does not require classification for skin and eye irritation according to EU CLP (EC 1272/2008) criteria.