Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Skin irritation/corrosion: Data waiving (study scientifically not necessary): According to column 2 of REACH Annex VII, the study does not need to be conducted because an acute toxicity study by the dermal route is available for the test substance and does not indicate skin irritation up to 2 000 mg/kg body weight (limit dose test).

Serious eye damage/eye irritation: Key study. Method according to OECD guideline 437, GLP study. The test item showed no effects on the cornea of the bovine eye. The test item does not require classification for eye irritation or serious eye damage.

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 an acute toxicity study by the dermal route does not indicate skin irritation up to the relevant limit dose level (2 000 mg/kg body weight)
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
According to column 2 of REACH Annex VII, the study does not need to be conducted because an acute toxicity study by the dermal route is available for the test substance and does not indicate skin irritation up to 2 000 mg/kg body weight (limit dose test).
Reason / purpose for cross-reference:
data waiving: supporting information
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 February 2020
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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
cattle
Strain:
other: Bos primigenius Taurus
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Müller Fleisch GmbH, Industriestraße 42, 75217 Birkenfeld, Germany.
- Number of animals: Not specified. 9 corneas received for the test.
- Characteristics of donor animals (e.g. age, sex, weight): Between 12 and 60 months old.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container within 1 hour and 5 minutes.
- Time interval prior to initiating testing: the test was performed on the day of the transport.
- indication of any existing defects or lesions in ocular tissue samples: None of the corneas received showed tissue damage; therefore, all corneas were used.
- Indication of any antibiotics used: Penicillin 100 U/mL, Streptomycin 100 µg/mL.
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): 750 µL.
- Concentration (if solution): undiluted

Duration of treatment / exposure:
10 min.
Duration of post- treatment incubation (in vitro):
2 hours.
Number of animals or in vitro replicates:
3 replicates.
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1ºC) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1ºC.
MEM: Minimum Essential Medium
cMEM (complete MEM): MEM supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum

QUALITY CHECK OF THE ISOLATED CORNEAS
Corneas which show any tissue damages or an opacity greater than seven opacity units should be discarded.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: yes, Hank’s Balanced Salt Solution (HBSS).

POSITIVE CONTROL USED: yes, Dimethylformamide, undiluted.

APPLICATION DOSE AND EXPOSURE TIME: 750 μL (test item, negative and positive controls), 10 min exposure.

TREATMENT METHOD: closed chamber method

POST-INCUBATION PERIOD: yes, 2 hours.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least three times or until no visual evidence of test chemical was observed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer BASF OP 3.0.
- Corneal permeability: passage of sodium fluorescein dye (optical density at 492 nm) measured with microtiter plate photometer (Anthos Reader 2010 Flexi).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: as indicated in the TG (see table below).

Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
0.07
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
(0.59)
Positive controls validity:
valid
Remarks:
(86.97)
Irritation parameter:
cornea opacity score
Run / experiment:
mean
Value:
0.05
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
(0.54)
Positive controls validity:
valid
Remarks:
(69.04)
Irritation parameter:
other: permeability
Run / experiment:
mean
Value:
0.001
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
(0.0037)
Positive controls validity:
valid
Remarks:
(1.1952)
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- A test is considered acceptable if: the positive control gives an IVIS that falls within two standard deviations of the current historical mean and negative/solvent controls gives values of opacity and permeability lower than upper limits for background values.
- Acceptance criteria met for negative control: yes, HBSS response resulted in opacity and permeability values that are less than established upper limits for negative control opacity (2.36) and permeability values (0.03)
- Acceptance criteria met for positive control: yes, IVIS score of dimethylformamide was 86.97 which falls between two standard deviations of the historical mean i.e. 56.56 – 139.37.

Table 1.1. Illuminance Values (Unit: LUX)

Parameter

Negative Control

Test Item

Positive Control

1. Rep.

2. Rep.

3. Rep.

1. Rep.

2. Rep.

3. Rep.

1. Rep.

2. Rep.

3. Rep.

(I) Measured values before exposure

1052

1045

1060

1014

1022

1024

1024

1033

1035

(I) Measured values after exposure

1038

1035

1044

998

1012

1009

382

414

382

Table 1.2. Opacity Values Negative Control

Parameter

Negative Control

1. Rep.

2. Rep.

3. Rep.

Opacity before exposure

2.44

2.72

2.12

Opacity after exposure

3.00

3.12

2.76

Opacity Difference

0.56

0.41

0.64

Mean Opacity Difference

0.54

Table 1.3. Opacity Values Test Item and Positive Control

Parameter

Test Item

Positive Control

1. Rep.

2. Rep.

3. Rep.

1. Rep.

2. Rep.

3. Rep.

Opacity before exposure

4.01

3.67

3.58

3.58

3.21

3.12

Opacity
after exposure

4.70

4.09

4.22

75.85

66.94

75.85

Opacity
Difference

0.70

0.43

0.64

72.27

63.73

72.72

Opacity
Difference
corrected

0.16

-0.11

0.10

71.73

63.20

72.19

Mean Opacity
Difference
corrected

0.05

69.04

Table 2.1. Optical density at 492 nm of Blank

Parameter

cMEM without phenol red

1. Measurement

0.040

2. Measurement

0.040

3. Measurement

0.035

Mean

0.038

Table 2.2. Optical density at 492 nm of Negative Control, Test Item and Positive Control

Parameter

Negative Control

Test Item

Positive Control

1. Rep.

2. Rep.

3. Rep.

1. Rep.

2. Rep.

3. Rep.

1.Rep.

2.Rep.

3.Rep.

1. Measurement

0.045

0.040

0.042

0.048

0.042

0.039

1.068

1.250

1.383

2. Measurement

0.043

0.040

0.041

0.049

0.043

0.037

1.095

1.243

1.380

3. Measurement

0.044

0.041

0.042

0.046

0.045

0.038

1.083

1.246

1.387

 

1. Measurement – blank

0.0067

0.0017

0.0037

0.0097

0.0037

0.0007

1.0297

1.2117

1.3447

2. Measurement – blank

0.0047

0.0017

0.0027

0.0107

0.0047

-0.0013

1.0567

1.2047

1.3417

3. Measurement – blank

0.0057

0.0027

0.0037

0.0077

0.0067

-0.0003

1.0447

1.2077

1.3487

Mean of each replicate

0.0057

0.0020

0.0033

0.0093

0.0050

-0.0003

1.0437

1.2080

1.3450

Mean of the 3 replicates

0.0037

--

--

Corrected

--

--

--

0.0057

0.0013

-0.0040

1.0400

1.2043

1.3413

Corrected mean of the 3 replicates

--

0.0010

1.1952

Table 3. IVIS

Test Group

IVIS

Mean IVIS

Relative Standard Deviation IVIS

Negative Control
HBSS

0.65

0.59

22.78%

0.44

0.69

Test Item
Ethyl-NENA

0.25

0.07

255.88%

-0.09

0.04

Positive Control
DMF undiluted

87.33

86.97

6.36%

81.26

92.31

Note: the high relative standard deviation of the IVIS of the test item is due to mathematical reasons, as the respective means are very small.

*IVIS = Mean Opacity value + (15 × Mean Permeability value)

Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
Under the conditions of this study, the test item showed no effects on the cornea of the bovine eye. The test item does not require classification for eye irritation or serious eye damage.


Executive summary:

An in vitro Bovine Corneal Opacity and Permeability (BCOP) study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 437 under GLP conditions. Three sets consisting of three corneas each were tested using the closed chamber method: the first set was the negative control and was treated with 750 μL Hank’s Balanced Salt Solution (HBSS); the second set was the positive control and was treated with 750 μL of dimethylformamide undiluted and the third set was treated with 750 μL of test item without dilution. The corneas were exposed for 10 min at 32ºC. After removal of the test item and 2 hours post-incubation, opacity of the corneas and fluorescein permeability were measured. Negative control showed No category (IVIS score 0.59) and resulted in opacity and permeability values that are less than the established upper limits for the background values. The positive control induced serious eye damage on the cornea (IVIS score 86.97) and was within two standard deviations of the current historical mean. The test item showed no effects on the cornea of the bovine eye. The calculated mean IVIS was 0.07. According to OECD Guideline 437, a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.

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

Additional information

Justification for classification or non-classification

Skin irritation/corrosion: Based on the available data, the substance is not classified for skin irritation according to CLP Regulation no. 1272/2008.

Serious eyes damage/eye irritation: Based on the available data, the substance is not classified for eye irritation according to CLP Regulation no. 1272/2008.

Categories Display