Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Skin irritation:

in vivo: irritating (OECD 404, BASF 2015a)

in vitro: not irritating (OECD 431/439 BASF 2015b)

Eye irritation:

in vivo: not irritating (OECD 405, BASF 2015c)

in vitro: not irritating (OECD 492 & OECD 437, BASF 2015d)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
yes
Remarks:
due to severe irritation, only two animals were used
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.: 0008924462
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, Netherlands
- Age at study initiation: Approx. 8-9 months
- Weight at study initiation: 3.58 kg – 4.09 kg
- Housing: Single housing
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Acclimatization for at least 5 days before application

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C +- 3
- Humidity (%): 30 – 70
- Air changes (per hr): ca. 10
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
other: untreated skin of the same animals
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL
Duration of treatment / exposure:
4 h
Observation period:
14 days
Number of animals:
2
Details on study design:
TEST SITE
- Area of exposure: dorsolateral part of the trunk
- % coverage: 2.5 cm x 2.5 cm)
- Type of wrap if used: The test patch was secured in position with a semi- occlusive dressing (The test item was covered with a test patch (Idealbinde, Pfälzische Verbandstoff-Fabrik, Kaiserslautern) and Fixomull® stretch (adhesive fleece), Beiersdorf AG)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: The test item was removed at the end of the exposure period with Lutrol®** and Lutrol® / water (1 : 1).

SCORING SYSTEM: according to OECD 404 guideline
Irritation parameter:
erythema score
Basis:
mean
Time point:
24/48/72 h
Score:
3.2
Reversibility:
not fully reversible within: 14 days
Remarks on result:
other: There was no indication of necrosis at study termination.
Irritation parameter:
edema score
Basis:
mean
Time point:
24/48/72 h
Score:
3.5
Reversibility:
not fully reversible within: 14 days
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
3.3
Max. score:
4
Reversibility:
not fully reversible within: 14 days
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
3
Max. score:
4
Reversibility:
not fully reversible within: 14 days
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
3.3
Max. score:
4
Reversibility:
not fully reversible within: 14 days
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
3.7
Max. score:
4
Reversibility:
not fully reversible within: 14 days

In the first animal well-defined erythema (grade 2) could be noted immediately after removal of the patch until hour 1. From hour 24 until hour 48, moderate erythema (grade 3) was seen in this animal. This finding increased to severe erythema (grade 4) at hour 72 and decreased to moderate erythema (grade 3) from day 7 until day 14. Slight edema (grade 2) was seen immediately after removal of the patch followed by moderate edema (grade 3) at hour 1 and severe edema (grade 4) at hour 24. This finding decreased to moderate edema (grade 3) from hour 48 to hour 72 and to very slight edema (grade 1) from day 7 until day 14. Both erythema and edema were noted beyond the application area over the entire observation period of 14 days. Moreover, blisters beyond the application area and a whitish discolored application area were seen in this animal at hour 48. Crusty as well as eczema-like skin lesions beyond the application area were seen at hour 72, while brownish discolored plaque-like incrustations and eczema-like skin lesions were seen on day 7. On day 14 scaling and incrustations were noted in this animal.

In the second animal well-defined erythema (grade 2) could be noted immediately after removal of the patch until hour 1. From hour 24 until day 14, moderate erythema (grade 3) was seen in this animal. Very slight edema (grade 1) was seen immediately after removal of the patch followed by slight edema (grade 2) at hour 1 and severe edema (grade 4) from hour 24 until hour 48. This finding decreased to moderate edema (grade 3) from hour 72 until day 7. Erythema and edema were noted beyond the application area. Moreover, blisters, bleeding and a whitish discolored application area beyond the application area were seen in this animal at hour 48. Brownish discolored crusty as well as eczema-like skin lesions beyond the application area were seen at hour 72, while

plaque-like incrustations and eczema-like skin lesions were seen on day 7. On day 14 scaling was noted in this animal, while incrustations had fallen off.

The cutaneous reactions were not reversible in the two animals within 14 days after removal of the patch. The first animal still revealed moderate erythema (grade 3) and very slight edema (grade 1) both beyond the application area, scaling and incrustations.

The second animal revealed moderate erythema (grade 3) beyond the application area and scaling. There was no indication of necrosis at study termination.

Mean scores over 24, 48 and 72 hours for each animal were 3.3 and 3.0 for erythema and 3.3 and 3.7 for edema.

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2014-06-10 to 2015-03-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
2013
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2013
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0008924462
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
According to guidelines and widely accepted test system.
Based on the results of ECVAM (European Center for Validation of Alternative Methods) funded validation studies, it was concluded by the ECVAM Scientific Advisory Committee that the EpiDerm™ human epidermis model is suitable to be used for distinguishing between corrosive and non-corrosive chemicals (ECVAM: ESAC statement on the application of the EpidermTM human skin model for skin corrosivity testing of 14-15 Mar 2000) as well as between irritant and non-irritant chemicals (ECVAM: ESAC statement on the scientific validity of in-vitro tests for skin irritation testing of 5 Nov 2008).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Incubation time: 3 h
- Wavelength: 570 nm (OD570)

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
not applicable


PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
- Irritant potential of the test material is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile PBS. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 30 µL

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5% (w/v) in water
Duration of treatment / exposure:
1 h
Duration of post-treatment incubation (if applicable):
42 +/- 4 h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
test item
Value:
68
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: no

Table 1 Individual and mean OD570 values, individual and mean viability values and standard deviations

Test substance

 

Tissue 1

Tissue 2

Tissue 3

Mean

Standard deviation

Negative Control (NC)

Mean OD570

2.160

2.206

2.317

2.228

 

Viability [% of NC]

97.0

99.0

104.0

100

3.62

Test item

Mean OD570

1.405

1.452

1.678

1.512

 

Viability [% of NC]

63.1

65.2

75.3

68

6.54

Positive Control (PC)

Mean OD570

0.082

0.081

0.072

0.079

 

Viability [% of NC]

3.7

3.7

3.2

4

0.25

Interpretation of results:
GHS criteria not met
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:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.: 0008924462
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, Netherlands
- Age at study initiation: Approx. 3-4 months
- Weight at study initiation: 2.63 kg – 3.02 kg
- Housing: Single housing
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Acclimatization for at least 5 days before application

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C +- 3
- Humidity (%): 30 – 70
- Air changes (per hr): ca. 10
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
unchanged (no vehicle)
Controls:
other: untreated eyes of the same animals
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
Duration of treatment / exposure:
24 hours
Observation period (in vivo):
21 days
Number of animals or in vitro replicates:
3
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): rinsed with 3 to 6 mL of hand warm tap water for 1 to 2 minutes using a syringe with a blunt probe.
- Time after start of exposure: after 24 hours of exposure

SCORING SYSTEM: according to OECD 405 guideline

TOOL USED TO ASSESS SCORE: hand-slit lamp
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Reversibility:
other: no effects on cornea observed
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Reversibility:
other: no effects on iris
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
24/48/72 h
Score:
1
Reversibility:
fully reversible within: 14 days
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
0.7
Reversibility:
fully reversible within: 14 days
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: no effects on cornea observed
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: no effects on cornea observed
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: no effects on cornea observed
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: no effects on iris
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: no effects on iris
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: no effects on iris
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 14 days
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 14 days
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 14 days
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 14 days
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: no chemosis
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 14 days

Cornea and iris were free of any signs of irritation during the whole observation period.

Slight conjunctival redness (grade 1) was noted in all animals from 1 hour up to hour 72 hours after application and persisted in one animal until study day 7.

Moderate conjunctival chemosis (grade 2) was noted in one animal 1 hour after application. This finding decreased to slight conjunctival chemosis (grade 1) and was noted from hour 24 until study day 7 in this animal. In another animal slight conjunctival chemosis (grade 1) was observed at hour 1 only, while in the third animal this finding was seen from hour 24 until study day 7.

In all animals severe discharge (grade 3) was noticed at hour 1. In one of these animals slight discharge (grade 1) was observed at hour 48.

Additional findings like injected scleral vessels in a circumscribed or circular area were noted in the animals from hour 1 until hour 48 at the latest. Hair loss was seen in two animals from day 7 until day 14 or on day 14, only. Scaling on the eye lids or under the eye was observed in one animal on study day 7, while in another animal this finding was noticed from study day 7 until study day 14. Incrustations on the eye lids or under the eye were observed in one animal on study day 7 and in the two other animals from study day 7 until study day 14. In one of these animals the incrustation, localized under the eye, was pea-sized on study day 7.

The ocular reactions were reversible in one animal within 7 and in two animals within 14 days after application.

Mean scores calculated for each animal over 24, 48 and 72 hours were 0.0, 0.0 and 0.0 for corneal opacity, 0.0, 0.0 and 0.0 for iris lesions, 1.0, 1.0 and 1.0 for redness of the conjunctiva and 1.0, 0.0 and 1.0 for chemosis.

Interpretation of results:
GHS criteria not met
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2014-01-10 to 2015-03-22
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)
Version / remarks:
2013
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0008924462
Details on test animals or tissues and environmental conditions:
BCOP
SOURCE OF COLLECTED EYES
- Source: Schlachthof Bensheim, Am Schlachthof 7-9, 64625 Bensheim, Germany
- Characteristics of donor animals: minimum 12 maximum 60 months old
- indication of any existing defects or lesions in ocular tissue samples: none
- Indication of any antibiotics used: none
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 µL
Duration of treatment / exposure:
10 min
Duration of post- treatment incubation (in vitro):
2 h
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagles’s MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour. After the equilibration period the medium in both chambers was replaced with fresh prewarmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that showed macroscopic tissue damage or an opacity value < 537 opacity units1 were discarded. The remaining corneas were then distributed into negative control, positive control and treatment groups.

QUALITY CHECK OF THE ISOLATED CORNEAS: see above

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: De-ionized water

POSITIVE CONTROL USED: 100% ethanol (PC 1) and 100% dimethylformamide (PC 2) for liquid test substances and surfactants

APPLICATION DOSE AND EXPOSURE TIME: 750 µL for 10 min

TREATMENT METHOD: open chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least three times with Eagle´s MEM

- POST-EXPOSURE INCUBATION: 2h

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: as indicated in the TG
Irritation parameter:
cornea opacity score
Run / experiment:
test item
Value:
0.1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein leakage
Run / experiment:
test item
Value:
0.003
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
test item
Value:
0.1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: no

Table 1 Opacity score, Permeability score, and In Vitro Irritancy score (IVIS) of the test substance, NC and PC

Test substance

Corrected Opacity Change Mean±SD

Corrected OD490 Mean±SD

IVIS per group mean±SD

Test item

0.1 ± 0.1

0.003 ± 0.003

0.1 ± 0.1

Negative control

2.0± 0.7

0.005 ± 0.004

2.1 ± 0.7

Positive control 1 (ethanol)

35.1± 3.8

1.509 ± 0.415

57.8 ± 6.0

Positive control (DMF)

105.2± 10.5

1.321 ± 0.809

125.0 ± 21.3

Interpretation of results:
GHS criteria not met
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2014-01-10 to 2015-03-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
2014a Draft Proposal for a new Test Guideline (EpiOcular)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0008924462
Details on test animals or tissues and environmental conditions:
Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
The EpiOcularTM model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinocytes used to model the human corneal epithelium (compare Figure 1). The EpiOcularTM tissues (surface 0.6 cm²) are cultured on especially prepared cell culture inserts (MILLICELLs, 10 mm diameter) and are commercially available as kits (EpiOcular™ 200), containing 24 tissues on shipping agarose.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 µL
Duration of treatment / exposure:
30 min
Duration of post- treatment incubation (in vitro):
2 h
Number of animals or in vitro replicates:
2
Details on study design:
Details of the test procedure used:

Two tissues were treated with each, the test substance, the PC and the NC. Due to the physical condition of the test substance the protocol for liquids was applied.

Pre-incubation of the tissues
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 1 mL assay medium and preconditioned in the incubator at 37 °C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 hours.

Pretreatment of the tissues
After the pre-incubation, the tissues were pre-treated with 20 μL of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes

Application of the test substance
Using a pipette, fifty microliter (50 μL) of the undiluted liquid test substance was applied covering the whole tissue surface. Control tissues were concurrently applied with 50 μL of sterile de-ionized water (NC) or with 50 μL of methyl acetate (PC). After application, the tissues were placed into the incubator until the total exposure time of 30 minutes was completed.

Removal of the test substance and postincubation period
To remove the test substance, the tissues were washed with sterile PBS. For this purpose the tissues were immersed and swiveled three times in each of three beakers filled with PBS. Washed tissues were immediately immersed into 12-well plates, pre-filled with 5 mL/well prewarmed medium (post-soak immersion) in order to remove residual test substance. After 12 minutes (liquids) of post-soak immersion, each tissue was dried on absorbent paper and transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium. In the 1st test run the washing procedure was intensified by careful wiping with a MEROCEL® sponge (Fritz Ruck Ophthalmologische Systeme GmbH, Germany) in order to remove as much test substance as possible. After the washing procedure the tissues were light greenish discolored. However, in the 2nd test run no wiping was necessary and no discoloration of the tissues was observed. Subsequently, the tissues were incubated at standard culture conditions for 2 hours (postincubation period).

MTT incubation
After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol at room temperature overnight or for at least 2 hours on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.


- RhCE tissue construct used: EpiOcular OCL-200 kit

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals:
The test substance was presumed to directly reduce MTT. Therefore, two freeze-killed control tissues each were treated with the test article and the negative control, in the same way as described in the following section.

- Acceptance criteria:
Assay acceptance criterion for the NC:
The absolute OD570 of the NC-tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is ≥ 1.0. The mean OD570 of the NC should not exceed 2.5.

Acceptance criteria for the PC:
Methyl acetate used as PC usually leads to a tissue viability of approx. 25%. A viability of < 60% is acceptable.

Assay acceptance criterion for tissue variability:
Two tissues were treated under the same conditions. A variability between the tissues is considered to be acceptable if the relative difference of the viability is ≤ 20%.
Irritation parameter:
other: % of NC viability mean
Run / experiment:
test item 1st run
Value:
60.6
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: % of NC viability mean
Run / experiment:
test item 2nd run
Value:
68.6
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Table 1: 1st and 2nd test run: Mean OD570 values, mean viability values and intertissue Variability

Test Substance

1strun

2ndrun

 

mean

Inter-tissue variability [%]

mean

Inter-tissue variability [%]

Negative Control [NC]

Mean OD570

2.432

 

2.008

 

Viability [% of NC]

100.0

0.4

100.0

8.3

Test item

Mean OD570

1.475

 

1.377

 

Viability [% of NC]

60.6

5.1

68.6

5.0

Positive Control

Mean OD570

0.496

 

0.651

 

Viability [% of NC]

20.4

3.3

32.4

9.3

Interpretation of results:
GHS criteria not met
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

In vivo

The potential of Triisooctylamine to cause acute dermal irritation or corrosivity was assessed in a study according to OECD 404 guideline and GLP (Bioassay, 2015) by a single topical application of an amount of 0.5 mL of the undiluted test item for 4 hours to the intact skin of two New Zealand White rabbits (stepwise procedure starting with one animal and supplementing one additional animal), using a patch of 2.5 cm x 2.5 cm, covered with semi-occlusive dressing. After removal of the patch the application area was washed off. The cutaneous reactions were assessed immediately after removal of the patch, approximately 1, 24, 48 and 72 hours after removal of the patch and in weekly intervals until day 14.

The following test item-related clinical observations were recorded during the course of the study:

- Well-defined to severe erythema (grade 2-4)

- Very slight to severe edema (grade 1-4)

- Erythema and edema beyond the application area

- Blisters beyond the application area

- Whitish discoloration of/beyond the application area

- Crusty as well as eczema-like skin lesions beyond the application area, partially brownish discolored

- Plaque-like incrustations, partially brownish discolored

- Eczema-like skin lesions

- Bleeding

- Scaling

- Incrustations

The cutaneous reactions were not reversible in the two animals within 14 days after removal of the patch. The first animal still revealed moderate erythema (grade 3) and very slight edema (grade 1) both beyond the application area, scaling and incrustations. The second animal revealed moderate erythema (grade 3) beyond the application area and scaling. There was no indication of necrosis at study termination. Mean scores over 24, 48 and 72 hours for each animal were 3.3 and 3.0 for erythema and 3.3 and 3.7 for edema. Considering the irreversibility of the described cutaneous reactions as well as the average score for irritation, Triisooctylamine shows a skin irritating potential under the test conditions chosen.

In vitro

The test item was assed regarding its potential for corrosive activity and skin irritation. Using the currently available methods a single in vitro assay may not always be sufficient to cover the full range of skin irritating/corrosion potential. Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT) according to OECD guidelines 431/439 and GLP. However, in the current case for Triisooctylamine the results derived with SIT alone were sufficient for a final assessment. Therefore further testing in SCT was waived. The potential of Triisooctylamine to cause dermal irritation was assessed by a single topical application of 30 μL of the undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDerm™). The irritation test was performed with three EpiDerm™ tissue samples, which were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the testsubstance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The EpiDerm™ skin irritation test showed the following results:

The test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 68%.

Based on the observed results it was concluded, that Triisooctylamine does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

 

Overall conclusion on skin irritation:

The test item was investigated for its skin irritating potential in both in vivo and in vitro test system. Results of the in vitro test predicted the test item to not be irritating to skin. In contrast, the test item turned out irritating in the in vivo study with rabbits. Therefore, the test item should be classified as being irritating according to current CLP Regulation.

Eye irritation

In vivo

The potential of Triisooctylamine to cause damage to the conjunctiva, iris or cornea was assessed in a study according to OECD 405 guideline and GLP (Bioassay, 2015) by a single ocular application of 0.1 mL of the undiluted test item to one eye of three New Zealand White rabbits (stepwise procedure starting with one animal and supplementing two additional animals). About, and not less than 24 hours after application the eye was rinsed with tap water.

The ocular reactions were assessed approximately 1, 24, 48 and 72 hours after application and in weekly intervals until study day 14 at the latest.

Additional eye examinations were performed at 24 and 48 h after application with the instillation of a fluorescein solution. Due to a negative finding at the 24 and 48 hour reading (no corneal lesions detectable with fluorescein) no further readings were performed with the aid of fluorescein.

The following test item-related clinical observations were recorded during the course of the study:

- Slight conjunctival redness (grade 1)

- Slight or moderate conjunctival chemosis (grade 1 or 2)

- Slight or severe discharge (grade 1 or 3)

Additional findings like injected scleral vessels in a circumscribed or circular area were noted in the animals from hour 1 until hour 48 at the latest. Hair loss was seen from day 7 until day 14 or on day 14 only. Scaling on the eye lid or under the eye was observed in one animal on study day 7 while in another animal this finding was noticed from study day 7 until study day 14. Incrustations on the eye lid or under the eye were observed in one animal on study day 7, and in the other two animals from study day 7 until study day 14. In one of these animals the incrustation, localized under the eye, was pea-sized on study day 7. (For details see table on page 21.)

The ocular reactions were reversible in one animal within 7 days and in two animals within 14 days after application.

Mean scores calculated for each animal over 24, 48 and 72 hours were 0.0, 0.0 and 0.0 for corneal opacity, 0.0, 0.0 and 0.0 for iris lesions, 1.0, 1.0 and 1.0 for redness of the conjunctiva and 1.0, 0.0 and 1.0 for chemosis.

Considering the described ocular reactions as well as the average score for irritation, Triisooctylamine does not show an eye irritating potential under the test conditions chosen.

In vitro

Possible eye irritating potential of Triisooctylamine was assessed in an in vitro Eye Irritation Turnkey Testing Strategy according to OECD 437 and OECD 492 test guidelines. Using the currently available methods a single in vitro assay may not always be sufficient to cover the full range of eye irritating potential. Therefore, two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular Eye Irritation Test.

BCOP

The potential of Triisooctylamine to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 μL of the undiluted test substance to the epithelial surface of isolated bovine corneas. Three corneas were treated with the test substance for 10 minutes followed by a 2-hours post-incubation period. In addition to the test substance a negative control (NC; de-ionized water) and two positive controls (PC 1: 100% ethanol and PC 2: dimethylformamid, DMF) were applied to three corneas each. Corneal opacity was measured quantitatively as the amount of light transmitted through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance. The results depicted in table 1 were obtained in the BCOP Test.

Table 1: Mean values for opacity, permeability and IVIS of the test substance, NC and PC

Test substance

Mean Opacity value

Mean Permeability Value

Mean in vitro irritancy score

Test item

0.1

0.003

0.1

NC

2.0

0.005

2.1

PC 1 (ethanol)

35.1

1.509

57.8

PC 2 (DMF)

105.2

1.321

125.0

 

 

EpiOcular

The potential of Triisooctylamine to cause ocular irritation was assessed by a single topical application of 50 μL of the undiluted test substance to a reconstructed three dimensional human cornea model (EpiOcular™). Two test runs were performed. Two EpiOcular™ tissue samples per test run were incubated with the test substance for 30 minutes followed by a 2-hour post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test substance treated epidermal tissues is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability. The EpiOcular™ eye irritation test showed the following results.

The test substance is not able to reduce MTT directly.

Due to inconclusive results after the 1st test run the study was repeated. In the 1st test run the mean viability of the test-substance treated tissues was 60.6%. Because the viabilities of the single tissues did not indicate a clear prediction (values for single tissues: 58.1 and 63.2%), a 2nd test run was performed. In the 2nd test run the mean viability of the test-substance treated tissues was 68.6% (values for single tissues: 66.1 and 71.1%). All acceptance criteria were met. Of the 4 valid tissues tested 3 tissues produced viabilities above the cut off for eye irritation and only 1 tissue showed a viability value (58.1%) below this cut off. The mean tissue viability of both test runs was calculated to be 64.6% with a standard deviation of 5.4%. Overall the results of both test runs did not indicate an irritation potential of the test substance.

The individual test results of the in vitro eye irritation turnkey testing strategy are summarized in table 2.

Table 2: Summary of individual test results and test strategy evaluation

Test method

Test Result

Test Evaluation

Evaluation Test Strategy

BCOP Test

The mean IVIS of the test-substance treated corneas was 0.1

Not identified as corrosive or severe irritant

Non-irritant

EpiOcular Test

Mean viability of the test-substance treated tissues of both runs was 64.6 %

Non-irritant

 

Based on the observed results for BCOP and EpiOcularTM Test it was concluded that Triisooctylamine does not show an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

Overall conclusion on eye irritation:

The test item was investigated for its eye irritating potential in both in vivo and in vitro test systems. Based on the in vitro tests outcome the test item is not considered irritating to the eye. This is further confirmed by the in vivo test. Therefore, the test item does not require classification as being irritating to the eye according to current CLP Regulation.


Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is considered to be classified as skin irritating Cat. 2 (H315: " Causes skin irritation") under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.

The test item is not classified as eye irritating under Regulation (EC) No 1272/2008, as amended for the tenth time in regulation (EU) No 2017/776.