Registration Dossier

Administrative data

Description of key information

According to REACH Annex VII column 2 no skin irritation/corrosion test has to be conducted as the substance is classified as acute dermal toxic cat. 1 (H310) according to CLP regulation Annex VI.

Based on a available in vitro test according to OECD 437 (BCOP) the substance has to be classified as eye irritant cat. 1 (H318).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because the substance is classified as acute toxicity by the dermal route (Category 1)
Endpoint conclusion
Endpoint conclusion:
no study available

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:
2016-08-09 to 2016-08-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
26 July 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
issued June 05, 2015
Specific details on test material used for the study:
SOURCE OF COLLECTED EYES
- Source: Slaughterhouse, Germany
- Storage, temperature and transport conditions of ocular tissue: transported in HBSS containig strep /Pen on ice to the
laboratory
- Time interval prior to initiating testing: corneae were isolated inmediatedly after arrival
of the eyes, corneae were directly used in the BCOP test on the same day
- indication of any existing defects or lesions in ocular tissue samples: eyes presenting defects were discarded



Species:
cattle
Strain:
other: cornea
Details on test animals or tissues and environmental conditions:
Invitro- Justification of the test method and considerations regarding applicability: The Bovine Corneal Opacity and Permeability (BCOP) test (OECD TG 437; TM B.47) is an in vitro test for the identification of substances inducing serious eye damage, recommended for use as part of a tiered-testing strategy for regulatory classification and labelling.- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live- :isolated corneas obtained as by -product from animals freshly slaughered. The fresh eyes were trasnported in HBSS containing Pen/Stre on ice to the laboratoriy. Inmediadely after arrival of the eyes the conrea preparation was initieated including:examination for defects and any defective eyes were discared.The coreas was excised leaving a 2 to 3 mm rim of sclera and stored in a petri dish containig HBSS and posterior mounted on corneal holders . The corneas (which are free of defects were incubated for one hour at 32 °C +-1
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.75 mL
- suspended with physiological saline 0.9% NaCl (B. Braun Melsungen lot no. 1406787) to gain a 20% concentration
Duration of post- treatment incubation (in vitro):
After 4 hours +- 5 minutes incubation at 32+-1 °C either test substance and the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of substance, the cormea was finally rinsed with complete RPMI (without phenol red)
Number of animals or in vitro replicates:
3 corneas for the test item, 3 corneas as negative controls treated with physiological saline 0.9%NaCL, 3 corneas as positive controls treated with imidazole 20% in physiological saline 0.9% Na Cl
Details on study design:
SELECTION AND PREPARATION OF CORNEAS: yes

QUALITY CHECK OF THE ISOLATED CORNEAS: yes

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: yes

POSITIVE CONTROL USED: yes

APPLICATION DOSE AND EXPOSURE TIME: 4 hours ± 5 minutes

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: three times with MEM


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacimeter (BASF-OP3.0, Duratec GmbH)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [Jenway 6405 UV/VIS spectrophotometry

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: yes
Irritation parameter:
in vitro irritation score
Value:
107.01
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current hystorical mean and therefore the assay is considered to be valid. See other information on results, table 4. The negative control responses result in opacity and permeability values less than the established upper limits of backround bovine cormeas treated with the negative control. All three corneas applicated with the test item showed an all over opacity of the tissue. The mean in vitro irritation score was calculated : 107.01
Visual observation: all 3 corneas treated with Scopolamine showed an allower opacity of the tissue.

Table1: Opacity

Cornea No.

Test item

Initial Opacity

Final Opacity

Change of Opacity Value

Corrected Opacity value

1

 

Negative Control

1.64

1.97

0.33

 

2

1.53

1.86

0.33

 

3

1.64

2.05

0.41

 

MV

1.60

1.96

0.36

 

1

 

Positive Control

2.58

102.31

99.73

99.37

2

2.54

97.64

95.10

94.74

3

2.85

86.34

83.49

83.13

MV

2.66

95.43

92.77

92.41

1

 

Test item

1.82

107.77

105.95

105. 59

2

0.56

66.06

65.50

65.14

3

0.81

54.77

53.97

53.61

MV

1.06

76.20

75.14

74.78

 

Table 2: Permeability

Cornea No.

Test item

OD490

Corrected

OD490 value

1

 

Negative Control

0.050

 

2

0.035

 

3

0.048

 

MV

0.044

 

1

 

Positive Control

3.355

3.311

2

3.495

3.451

3

3.260

3.216

MV

3.37

3.326

1

 

Test item

1.830

1.786

2

2.140

2.096

3

2.610

2.566

MV

2.193

2.149

 

Table 3: In Vitro Irritation Score

Cornea No.

Test item

OD490

Corrected

OD490 value

IVIS

1

 

Negative Control

0.33

0.050

 

2

0.33

0.035

 

3

0.41

0.048

 

MV

0.36

0.044

1.02

1

 

Positive Control

99.37

3.311

 

2

94.74

3.451

 

3

83.13

3.216

 

MV

92.41

3.326

142.30

1

 

Test item

105. 59

1.786

 

2

65.14

2.096

 

3

53.61

2.566

 

MV

74.78

2.149

107.01

 

Table 4: Historical mean In Vitro Irritation Score of the Positive Control

 

IVIS Positive Control

Mean value (MV)

125.73

Standard Deviation (SD)

17.05

MV-2xSD

91.64

MV-2xSD

159.83

Number of replicates providing Historical Mean: 15

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
All three cormeas applicated with the test item showed an all over opacity of the tissue. The mean in vitro irritation score was calculated to be 107.01. According to the evaluation criteria of the in vitro BCOP test (OECD 437), the test item was classified into UN GHS category 1.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

According to CLP regulation Annex VI column the substance is classified as acute dermal toxic cat. 1 (H310) hence a skin irritation study is not needed.

Based on a available test according to OECD 437 (BCOP) the substance has to be classified as eye irritant cat. 1( H318).