Registration Dossier

Administrative data

Description of key information

Skin irritation (OECD 404): not irritating

Eye irritation (OECD 437) not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Mar 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
1981
Deviations:
yes
Remarks:
4 concentrations of the test substance were tested on each animal
GLP compliance:
yes
Species:
rabbit
Strain:
Himalayan
Remarks:
Chbb:HM
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 2.4 - 2.8 kg
- Housing: Animals were housed individually in PPL cages (45 cm x 55 cm) with perforated floor.
- Diet: Altromin 2123, pelleted complete rabbit diet (Chr. Petersen, Ringsted, Denmark), ad libitum
- Water: fresh water, ad libitum
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 6
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
other: diethyl phthalate
Controls:
other: adjacent areas of skin treated with vehicle served as control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.5 mL
- Concentration: 1, 5, 25 and 100%
Duration of treatment / exposure:
4 h
Observation period:
72 h; Since all observed effects had completely reversed at the 48-h reading, the study was discontinued after the 72 h reading.
Reading time points: 30 min and 24, 48 and 72 h
Number of animals:
4
Details on study design:
TEST SITE
- Area of exposure: area on the back (10 cm x 10 cm) divided into two anterior and two posterior treatment sites and one test site at the center.
- Type of wrap: The test substance of one of the test concentrations or the vehicle was applied to the skin and covered with gauze packs (2.5 cm x 2.5 cm). The gauze packs were secured with a cross of 1 cm wide adhesive tape and fixed with Scanpor tape (7.5 cm width).

REMOVAL OF TEST SUBSTANCE
- Washing: The skin was cleaned of residual test substance with soap and lukewarm water.
- Time after start of exposure: 4 h

SCORING SYSTEM: Draize scoring system
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 h
Remarks on result:
other: 100%
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 h
Remarks on result:
other: 100%
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: 100%
Irritation parameter:
erythema score
Basis:
animal #4
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: 100%
Irritation parameter:
erythema score
Basis:
mean
Remarks:
of 4 animals
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: at test substance concentration of 1, 5 and 25%
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: at test substance concentration of 1, 5, 25 and 100%
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: at test substance concentration of 1, 5, 25 and 100%
Irritation parameter:
edema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: at test substance concentration of 1, 5, 25 and 100%
Irritation parameter:
edema score
Basis:
animal #4
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: at test substance concentration of 1, 5, 25 and 100%

Table 1. Results of the skin irritation study.

Rabbit no.

Test concentration

Erythema

Individual mean values

Edema

Individual mean values

½ h

24 h

48 h

72 h

½ h

24 h

48 h

72 h

1

AL

100%

2

1

0

0

0.33

0

0

0

0

0

2

PL

100%

2

1

0

0

0.33

0

0

0

0

0

3

PL

100%

1

0

0

0

0

0

0

0

0

0

4

PR

100%

0

0

0

0

0

0

0

0

0

0

AL: Anterior left treatment site

PL: Posterior left treatment site

RP: Posterior right treatment site

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of this skin irritation study in rabbits the test substance was not irritating to the skin.
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:
24 Nov 2017
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:
Jul 2013
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
Commission Regulation (EU) No 1152/2010 of 09 Dec 2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Odenwald Schlachthof Betriebs GmbH, Brensbach, Germany
- Characteristics of donor animals: at least 9 month old
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were transported in Hank's Buffered Salt Solution (HBSS) containing 1% (v/v) Penicillin/Streptomycin (100 units/mL, respectively).
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes and directly used in the BCOP test.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.75 mL

POSITIVE CONTROL
- Amount applied: 0.75 mL

NEGATIVE CONTROL
- Amount applied: 0.75 mL
Duration of treatment / exposure:
10 min at 32 ± 1 °C
Duration of post- treatment incubation (in vitro):
2 h in a vertical position
Number of animals or in vitro replicates:
triplicates for each treatment and control groups
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. Each cornea was mounted in a specially designed cornea holder.

QUALITY CHECK OF THE ISOLATED CORNEAS
At the end of the equilibration period, the basal opacity was determined (t0). Each cornea with a value of the basal opacity >7 was discarded.

TREATMENT METHOD
Closed chamber:
The cornea holder consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on the top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. After equilibration for about 1 hour, the anterior compartment received the test substance or the controls on the surface of the corneae. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath for 10 minutes.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 1; The test substance was rinsed off from the application side with saline.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer (OP_KiT opacitometer, Electro Design, France).
- Corneal permeability: The passage of sodium fluorescein dye was measured with the aid of a microplate reader (Versamax Molecular Devices) at 490 nm (OD490).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) , IVIS = opacity value + (15x OD490 value)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as serious eye damage and labelled Category 1 according to CLP/EPS/GHS.
Test substance with an IVIS < 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; < 55 no prediction can be made.
Irritation parameter:
in vitro irritation score
Remarks:
mean value of 3 corneae
Run / experiment:
10 min exposure
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control resulted in opacity and permeability values that were less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.
- Acceptance criteria met for postive control: The positive control resulted in an IVIS which was within two standard deviations of the current historical mean.

Table 2. Results after 10 min treatment time

 

 

Test Group

 

Opacity value = Difference (t130-t0) of Opacity

 

Permeability at 490 nm (OD490)

 

 

IVIS

 

 

Mean IVIS

 

Proposed

in vitro Irritancy Score

 

 

Mean

 

Mean

 

 

 

 

 

Negative Control

1

 

 

 

0.67

0.061

 

 

 

0.079

1.92

 

 

 

1.86

 

 

 

No Category

1

0.068

2.02

0

0.109

1.64

 

 

Positive Control

56.33*

1.703*

81.87

 

 

 

87.23

 

 

 

Category 1

61.33*

1.652*

86.11

73.33*

1.359*

93.71

 

Test subsatance

0.33*

-0.027*

-0.08

 

 

 

0.00**

 

 

 

No Category

0.33*

0.014*

0.54

-0.67*

-0.020*

-0.97

*corrected values

**negative values are set to zero

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
Under conditions of the Bovine Corneal Opacity and Permeability Test (BCOP) the test substance was not irritating to the eye. Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 0.00.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin

The skin irritation potential of the test substance was determined by two primary skin irritation/corrosion studies in the rabbit.

 

In a primary skin/corrosion study according to OECD Guideline 404 and in compliance with GLP 0.5 mL undiluted test substance and test substance at concentrations of 1, 5 and 25% in diethyl phthalate were applied sequentially to the clipped skin of 4 female Chbb:HM rabbits under semi-occlusive conditions for 4 h (1989). Scoring of skin reactions (erythema and edema) was performed 30 min and 24, 48 and 72 h after removal of the patch. No skin irritation was caused by 4 hours exposure to the test substance at concentrations of 1, 5 and 25%. Exposure to the undiluted test substance resulted in well-defined erythema (grade 2) in two animals and very slight erythema (grade 1) at the 30 min reading time point. The erythema had resolved within 24 and 48 h, respectively. No edema was found after application of the undiluted test substance. The irritation scores over 24, 48, and 72 h for the 4 animals were 0.33, 0.33, 0.0 and 0.0, respectively for the four animals. Based on the results, the test substance was not irritating to the skin under the conditions of the test.

In a second primary skin/corrosion study according to OECD Guideline 404 and in compliance with GLP 0.5 mL undiluted test substance and test substance at concentrations of 1, 5 and 25% in ethanol was applied sequentially to the clipped skin of 4 female Chbb:HM rabbits under semi-occlusive conditions for 4 h (Supporting, 1989). Scoring of skin reactions (erythema and edema) was performed 30 min and 24, 48 and 72 h after removal of the patch. No skin irritation was caused by 4 hours exposure to the test substance at concentrations of 1, 5 and 25%. Exposure to the undiluted test substance resulted in very slight erythema (grade 1) in one animal at the 24 h reading time point and very slight erythema (grade 1) in three animals at the 24, 48 and 72 h reading time points. Very slight edema was found in one animal at the 24 h reading time point. The edema had resolved within 48 h. The irritation scores over 24, 48, and 72 h were 1.0, 0.33, 1.0, 1.0 for erythema and 0.33, 0.0, 0.0, and 0.0 for edema for the four animals. Since observation period was terminated at 72 h, reversibility of erythema could not be evaluated and therefore this study was not used for classification purposes. As no scores greater than 1 were observed at any reading during the study period, the test item was assessed as not skin irritating by the study authors according to the relevant regulations at the time and the study was terminated before reversibility had been demonstrated. Despite this deficiency, the study can still be used to support the result of the key study as only very slight skin irritation was observed.

Eye

The eye irritation potential of the test substance was determined in a bovine corneal opacity and permeability (BCOP) test according to OECD Guideline 437 and in compliance with GLP (2017). After a first opacity measurement of the fresh bovine corneae, the neat test substance was applied directly to the epithelial surface of three cattle corneae in an incubation chamber in horizontal position for 10 min at 32 ± 1 °C. After the incubation phase the test substance was rinsed from the corneae with saline. Further, the corneae were incubated for another 2 hours at 32 ± 1 °C in a vertical position, while the anterior chamber contained incubation medium as well. Afterwards, opacity was measured a second time. In addition, the permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C. With the negative control, 0.9% (w/v) NaCl solution in deionised water, neither an increase of opacity nor permeability of the corneae could be observed. The mean in vitro irritation score (IVIS) of the negative control was 1.86. The positive control, 2-ethoxyethanol, showed clear opacity and distinctive permeability of the corneae fulfilling the criteria for classification as severe irritating/corrosive. The mean IVIS of the positive control was 87.23. All three values of the negative and positive controls, respectively, were within the range of historical data of the test facility. Relative to the negative control, the test substance did not cause an increase of the corneal opacity and permeability. The calculated mean IVIS was 0.00 (threshold for serious eye damage: IVIS > 55). Based on the results of this test, the test substance is considered to be not irritating to the eye.

Justification for classification or non-classification

The available data on skin and eye irritation do not meet criteria for classification according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.