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Description of key information

Skin irritation

In an in vitro skin irritation test using a human skin model ( EPISKIN Standard Model), the influence of the test substance on the viability of human skin was tested. The test substance was applied directly to 0.38 cm2 cultured skin (25 µl). After 15 minutes, the substance was removed and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 19% whereas the test substance showed a cell viability of 33%. Since the mean relative tissue viability after exposure to the test substance was below 50%, it can be concluded that the test substance is irritating in the in vitro skin irritation test.

Eye Irritation

The registration substance was tested for its ocular irritation properties by exposing isolated bovine cornea for 10 minutes to the test substance according to OECD 437. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range and the mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The registration substance did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.2 after 10 minutes of treatment. It is concluded that the registration substance is not irritant or corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

In an eye irritation study with rabbits, performed according to OECD and OPPTS test guidelines, the eyes of 3 rabbits were exposed to 0.1 mL of the test substance. Conjunctival irritation was noted for all 3 rabbits. The only grade 2 finding was observed for one male at 24 hours post-dosing. All other conjunctival irritation was graded as 1 and subsided by day 10. Therefore, the substance is considered to be not an irritant for the eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation / corrosion
Remarks:
other: in vitro (validated and accepted)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
other: EU method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test), adopted 24 August 2009
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline no. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, adopted 22 July 2010
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): EXP1200078
- Physical state: dark brown viscous liquid
- Storage condition of test material: at room temperature in the dark

- Analytical purity: 100%
- Lot/batch No.: E00275-350
- Expiration date of the lot/batch: 31 Dec 2013
- Stability at higher temperatures: yes, maximum temperature 60 °C; maximum duration 1 hour
Species:
human
Details on test animals and environmental conditions:
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 36.9-37.5
- Humidity (%): 74-97
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amounts applied: 25 µl

NEGATIVE CONTOL:
- Amount applied: 25 µl Phosphate buffered saline

POSITIVE CONTROL
- Amount applied: 25 µl
- Concentration: 5% (aq) sodium dodecyl sulphate
Duration of treatment / exposure:
15 minutes
Observation period:
not applicable
Number of animals:
not applicable
Details on study design:
TEST SITE
- EPISKIN Standard ModelTM(EPISKIN-SMTM, 0.38 cm2, Lot no.: 13-EKIN-009).This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded in 12-well plates on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days , which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

REMOVAL OF TEST SUBSTANCE
- Washing: phosphate buffered saline
- Time after start of exposure: 15 minutes

POST INCUBATION PERIOD
- 42 hours

SCORING SYSTEM:
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.
Irritation / corrosion parameter:
% tissue viability
Value:
33
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The relative mean tissue viability obtained after 15 minutes treatment with EXP1200078 compared to the negative control tissues was 33%. Although the test substance shows interaction with MTT, which can result in a false negative (non-irritant) conclusion, the outcome of the mean relative tissue viability for EXP1200078 was below 50% after 15 minutes treatment. Therefore the substance is considered to be irritant.

The test substance was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because a colour change was observed it was concluded that the test substance did interact with MTT. In addition to the normal procedure, three killed tissues treated with test substance and three killed non treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by EXP1200078 was 49% of the negative control tissues.

The non-specific MTT (NSMTT) was outside the acceptability criteria of the assay. A high NSMTT indicates that the test substance shows a high interaction with MTT. When a high response of MTT is observed, a high OD570 will be the consequence. However, although the test substance interacts with MTT, the test substance showed a very low OD570 without correction for the NSMTT. Correcting for the NSMTT would result in negative values. Therefore, the NSMTT was not taken into account for the determination of the viability of the test substance treated skin tissues.

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The in vitro skin irritation test was conducted according to OECD 439 and GLP principles.
It is concluded that this test is valid and that the test substance is irritating in the in vitro skin irritation test.
Executive summary:

In an in vitro skin irritation test using a human skin model ( EPISKIN Standard Model), the influence of the test substance on the viability of human skin was tested. The test substance was applied directly to 0.38 cm2 cultured skin (25 µl). After 15 minutes, the substance was removed and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 19% whereas the test substance showed a cell viability of 33%. Since the mean relative tissue viability after exposure to the test substance was below 50%, it can be concluded that the test substance is irritating in the in vitro skin irritation test.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 2013 - February 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
(2012)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Version / remarks:
(1998)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): EXP1200078
- Physical state: dark brown viscous liquid
- Storage condition of test material: at room temperature in the dark

- Analytical purity: 100%
- Lot/batch No.: E00275-350
- Expiration date of the lot/batch:End-2015
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
- Source: Covance Research Products, Inc., Greenfield, IN, USA
- Age at study initiation: Animals used within the study were approx 9 months old.
- Weight at study initiation: Body weights were 3321-3459 g.
- Housing: Individually housed in stainless steel cages.
- Diet: The basal diet for rabbits (PMI Nutrition International, LLC, Certified Rabbit HF LabDiet® 5325)was provided at approx 150 g/day.
- Water: Free access to tap water.
- Acclimation period: At least 5days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.5-18.7
- Humidity (%): 45.4-53.1
- Air changes (per hr): approx 10
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
unchanged (no vehicle)
Controls:
other: One eye of each animal remained untreated and served as the reference control.
Amount / concentration applied:
TEST MATERIAL- Amount applied: 0.1 mL (liquid)
Duration of treatment / exposure:
Single instillation on Day 1.
Observation period (in vivo):
The eyes of each animal were examined approximately 1, 24, 48 and 72 hours after dosing, and on days 4, 7, 10 if irritation persisted.
Number of animals or in vitro replicates:
3 males
Details on study design:
TREATMENT
Approximately 60 minutes prior to test substance application, buprenorphine (0.01 mg/kg) was administered by subcutaneous injection (SC) to provide a therapeutic level of systemic analgesia.
Approximately 5 minutes prior to test substance application, one drop of a topical ocular anesthetic (0.5% proparacaine hydrochloride) was applied to each eye. The test substance (0.1 mL) was placed directly into the cupped lower conjunctival sac of each rabbit’s right (test) eye. The eyelid was held closed for approximately 1 second after instillation and then released. The left eye of each animal was manipulated in an identical manner to simulate the dosing of the right eye. Following test substance administration, no additional topical anesthetics were applied in order to avoid interference with the study.
Initially, a single animal was dosed. Based on the results 2 additional rabbits were exposed.
Approximately 8 hours after test substance administration, buprenorphine (0.01 mg/kg) SC and meloxicam (0.5 mg/kg) SC were administered to provide a continued therapeutic level of systemic analgesia. Following the initial 8-hour post-dosing treatment, buprenorphine (0.01 mg/kg) SC was administered every twelve (12) hours, in conjunction with meloxicam (0.5 mg/kg) SC every 24 hours, until positive ocular lesions/irritation resolved and no clinical signs of pain and distress were detected.

REMOVAL OF TEST SUBSTANCE
-Washing: No

OBSERVATIONS (beschrijf beperkt)
- Mortality/Viability: Twice daily.
- Toxicity: At least once daily.
- Body Weight: Obtained on study day 0 (initiation) and at each rabbit’s termination from the study (study day 7 or 10).
- Necropsy: the rabbits were euthanized by intravenous injection of sodium pentobarbital and discarded.
- Irritation: prior to initiation of dosing. The pre-initiation examination included the use of sodium fluorescein and a direct ophthalmoscope for detection of corneal abnormalities. Only rabbits with no pre existing ocular abnormalities were assigned to study. Both eyes of all rabbits were examined macroscopically for ocular irritation in accordance with the method of Draize at approximately 1, 24, 48 and 72 hours, and days 4, 7, 10 after instillation of the test substance when irritation persisted. The irritation scores and a description of all other (local) effects were recorded.The irritation was assessed according to OECD 405.
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1.33
Max. score:
3
Reversibility:
fully reversible within: 10
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.3
Max. score:
4
Reversibility:
fully reversible within: 2 days
Irritant / corrosive response data:
Conjunctival irritation was noted for all 3 rabbits. The only grade 2 finding was observed for one male at 24 hours post-dosing. All other conjunctival irritation was graded as 1 and subsided by study day 10.
The left (control) eyes were free of evidence of ocular irritation and other findings for the duration of the study.
Other effects:
One animal had light brown staining of hair surrounding the eye. The other 2 animals had dried light brown material around eye.

There were no deaths during the study, nor test substane related clinical observations.
There were no remarkable body weight changes noted during the study. One rabbit had a very slight body weight loss (0.8%) from study days 0 to 7. However, body weight losses are commonly associated with animals that are on a restricted diet and can be enhanced by the use of systemic analgesics.
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In an eye irritation study with rabbits, performed according to OECD test guidelines, slight irritation was observed, which disappeared by day 10.
Executive summary:

In an eye irritation study with rabbits, performed according to OECD and OPPTS test guidelines, the eyes of 3 rabbits were exposed to 0.1 mL of the test substance. Conjunctival irritation was noted for all 3 rabbits. The only grade 2 finding was observed for one male at 24 hours post-dosing. All other conjunctival irritation was graded as 1 and subsided by day 10. Therefore, the substance is considered to be not an irritant for the eyes.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Feb 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
other: OECD guideline 437 “Bovine corneal opacity and permeability (BCOP) test method for identifying ocular corrosives and severe irritants” (07 Sept 2009)
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Method B.47 “Bovine corneal opacity and permeability method for identifying ocular corrosives and severe irritants (09 Dec2010)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): EXP1200078
- Physical state: dark brown viscous liquid
- Storage condition of test material: at room temperature in the dark

- Analytical purity: 100%
- Lot/batch No.: E00275-350
- Expiration date of the lot/batch: 31 Dec 2013
- Stability at higher temperatures: yes, maximum temperature 60 °C; maximum duration 1 hour
Species:
other: in vitro; bovine eyes
Details on test animals or tissues and environmental conditions:
Source: slaughterhouse

Temperature (°C): 32 ± 1
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µl per cornea

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µl of physiological saline per cornea

POSITIVE CONTROL
Amount(s) applied (volume or weight with unit): 750 µl per cornea
Concentration (if solution): 10% (w/v) benzalkonium chloride


Duration of treatment / exposure:
10 minutes
Observation period (in vivo):
not applicable
Number of animals or in vitro replicates:
not applicable
Details on study design:
TEST SITE
- Isolated bovine cornea

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 10 minutes

SCORING SYSTEM:
- After exposure the cornea is thoroughly rinsed to remove the test substance and incubated for another 2 hours in Eagle's Minimum Essential Medium followed by immediate opacity measurement and permeability evaluation of the cornea.

- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:

In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).


TOOL USED TO ASSESS SCORE:
- opacitometer and microplate reader

DATA EVALUATION:
A test substance that induces an IVIS = 55.1 is defined as a corrosive or severe irritant
Irritation parameter:
in vitro irritation score
Value:
0.2
Vehicle controls validity:
not examined
Negative controls validity:
valid
Remarks:
Physiological saline; score was -1.0
Positive controls validity:
valid
Remarks:
Benzalkonium chloride, 10% (w/v); score was 125
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein retention score
Value:
2.78
Vehicle controls validity:
not examined
Negative controls validity:
valid
Remarks:
Physiological saline; score was 0.000
Positive controls validity:
valid
Remarks:
Benzalkonium chloride, 10% (w/v)
Irritation parameter:
cornea opacity score
Value:
0
Vehicle controls validity:
not examined
Negative controls validity:
valid
Remarks:
Physiological saline; score was -1
Positive controls validity:
valid
Remarks:
Benzalkonium chloride, 10% (w/v); score was 83
Remarks on result:
no indication of irritation
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
EXP1200078 is not irritant or corrosive in the Bovine Corneal Opacity and Permeability test.

Executive summary:

EXP1200078 was tested for its ocular irritation properties by exposing isolated bovine cornea for 10 minutes to the test substance according to OECD 437.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range and the mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

EXP1200078 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.2 after 10 minutes of treatment.

It is concluded that EXP1200078 is not irritant or corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The inhalation study need not be conducted because exposure of humans via inhalation is not likely taking into account the vapor pressure of the substance and/or the possibility of exposure to aerosols, particles or droplets of an inhalable size.

Justification for classification or non-classification

In accordance to Regulation (EC) No 1272/2008 the registration substance is classified as non-irritating for eyes based on the results of the ex vivo and in vivo studies, but a category 2 skin irritant based on data from the in vitro study.