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Administrative data

Description of key information

skin irritation/corrosion (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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
adopted in Apr 2002
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Version / remarks:
2004/73/EC
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Behörde für Arbeit, Gesundheit und Soziales, Hamburg, Germany
Species:
rabbit
Strain:
Himalayan
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: approx. 5.5 - 6.5 months
- Weight at study initiation: 2.2 - 2.5 kg
- Housing: animals were kept singly in cages (400 mm x 500 mm x 370 mm) before and after exposure, singly in restrainers during exposure
- Diet: ssniff K-H V233 (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.5 mL
Duration of treatment / exposure:
4 h
Observation period:
14 days
Reading time points: 1, 24, 48 and 72 h and 4 to 14 days
Number of animals:
3 males
Details on study design:
TEST SITE
- Area of exposure: 6 cm² on the dorsal area of the trunk
- Type of wrap: The treated skin was covered with a gauze patch, which was held in place with a non-irritating tape.

REMOVAL OF TEST SUBSTANCE
- 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:
1
Max. score:
4
Reversibility:
fully reversible within: 12 days
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 14 days
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 12 days
Irritation parameter:
edema score
Basis:
mean
Remarks:
of 3 animals
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritant / corrosive response data:
An erythema (grade 1) was observed in animal #1 and #2 from 24 h to 11 and 13 days, respectively, after patch removal. An erythema (grade 1) was observed in animal #3 from 1 h to 11 days after patch removal.
Other effects:
Induration of the skin was observed in animal #1 7 to 9 days after patch removal.
There were no systemic intolerance reactions.

Table 1. Results of the skin irritation study.

Observation time

Rabbit no.

1

2

3

Erythema

Edema

Erythema

Edema

Erythema

Edema

1 h

0

0

0

0

1

0

24 h

1

0

1

0

1

0

48 h

1

0

1

0

1

0

72 h

1

0

1

0

1

0

Mean value
24 + 48 + 72 h

1.00

0

1.00

0

1.00

0

4 days

1

0

1

0

1

0

5 days

1

0

1

0

1

0

6 days

1

0

1

0

1

0

7 days

1

0#

1

0

1

0

8 days

1

0#

1

0

1

0

9 days

1

0#

1

0

1

0

10 days

1

0

1

0

1

0

11 days

1

0

1

0

1

0

12 days

0

0

1

0

0

0

13 days

-

-

1

0

- 

 -

14 days

-

-

0

0

- 

 -
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.
Executive summary:

The skin irritation potential of the test substance was determined by an in vivo skin irritation test in rabbits according to OECD Guideline 404 and in compliance with GLP (2004). A dose of 0.5 mL of the test substance was applied to the skin of three rabbits under semi-occlusive conditions for 4 hours. After the exposure period the patch was removed and the skin sites were evaluated. Scores were taken 60 minutes, 24, 48, 72 hours and 4 to 14 days after patch removal. An erythema (grade 1) was observed in two animals (no. 1 and 2) from 24 h to 11 and 13 days, respectively, after patch removal. An erythema (grade 1) was observed in animal no. 3 from 1 h to 11 days after patch removal. In addition, induration of the skin was observed in animal no. 1 from Day 7 to 9. No systemic intolerance reactions were observed. Based on the results, the test substance was not irritating to the skin under the conditions of the test.

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:
6 Feb 2015
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)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Species:
other: cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Schlachthof Bensheim, Bensheim, 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) with streptomycin / penicillin at ambient temperature.
- 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 on the same day.
- 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
- Applied volume: 0.75 mL

POSITIVE SUBSTANCE
- Applied volume: 0.75 mL

NEGATIVE CONTROL
- Applied volume: 0.75 mL
Duration of treatment / exposure:
10 min at 32 ± 1 °C
Duration of post- treatment incubation (in vitro):
2 h
Number of animals or in vitro replicates:
triplicates for each treatment and control group
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 a basal opacity >7 was discarded.

TREATMENT METHOD:
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.

NUMBER OF REPLICATES: 3 corneae per test group

REMOVAL OF TEST SUBSTANCE:
The test substance was rinsed off from the application side with saline.
- POST-EXPOSURE INCUBATION: 2 h in a vertical position

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 1according 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
Run / experiment:
mean value of 3 corneae
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritant / corrosive response data:
With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 0.38).
The positive control (2-ethoxyethanol) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 77.72) corresponding to a classification as serious eye damaging.
Relative to the negative control, the test item did not cause an increase of the corneal opacity or permeability (mean IVIS = 0.0) and thus the test substance is not categorized.

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 positive 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 incubation time.

Test group

Opacity value =
Difference (t130-t0) of Opacity

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

 

Mean

 

Mean

 

 

Negative

control

0

-0.33

0.049

0.048

0.74

0.38

0

0.048

0.72

-1

0.046

-0.31

Positive

control

67.33*

0.950*

81.59

77.72

70.33*

0.754*

81.65

56.33*

0.906*

69.93

Test substance

0.33*

0.001*

0.35

0.00

-0.67*

-0.001*

-0.68

-0.67*

-0.005*

-0.74

*: corrected values

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 the BCOP assay 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.
Executive summary:

The eye irritation potential of the test substance was determined in a bovine corneal opacity and permeability test (BCOP test) according to OECD Guideline 437 and in compliance with GLP (2015). 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. Further, the corneae were incubated for another 120 min 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 min at 32 ± 1 °C. The results of the opacity and permeability measurement of the test substance were used to calculate an in vitro irritation score (IVIS) of 0.00. With the negative control saline neither an increase of opacity nor permeability of the corneae could be served. The mean IVIS of the negative control was 0.38. The mean IVIS of the positive control (2-ethoxyethanol) was 77.72. Based on the results, the test substance was not irritating to the eye under the conditions of the test.

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 an in vivo skin irritation test in rabbits according to OECD Guideline 404 and in compliance with GLP (2004). A dose of 0.5 mL of the test substance was applied to the skin of three rabbits under semi-occlusive conditions for 4 hours. After the exposure period the patch was removed and the skin sites were evaluated. Scores were taken 60 minutes, 24, 48, 72 hours and 4 to 14 days after patch removal. An erythema (grade 1) was observed in two animals (no. 1 and 2) from 24 h to 11 and 13 days, respectively, after patch removal. An erythema (grade 1) was observed in animal no. 3 from 1 h to 11 days after patch removal. In addition, induration of the skin was observed in animal no. 1 from Day 7 to 9. No systemic intolerance reactions were observed. Based on the results, the test substance was not irritating to the skin under the conditions of the test.

Eye

The eye irritation potential of the test substance was determined in a bovine corneal opacity and permeability test (BCOP test) according to OECD Guideline 437 and in compliance with GLP (2015). 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. Further, the corneae were incubated for another 120 min 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 min at 32 ± 1 °C. The results of the opacity and permeability measurement of the test substance were used to calculate an in vitro irritation score (IVIS) of 0.00. With the negative control saline neither an increase of opacity nor permeability of the corneae could be served. The mean IVIS of the negative control was 0.38. The mean IVIS of the positive control (2-ethoxyethanol) was 77.72. Based on the results, the test substance was not irritating to the eye under the conditions of the test.

Justification for classification or non-classification

The available data on skin and eye irritation of the test substance do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.