Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Skin irritation/corrosion

In EPISKIN model (Harlan 2013), reconstructed human skin tissues in triplicate were treated with 30 uL of the test substance during 15 minutes. Optical density of extracted MTT solutions was measured at 540 nm. Viability was expressed as the corrected percentage MTT conversion versus negative control. The viability was 109.2%, which is indicative for non-irritant substances. The test substance is non-irritant in the EPISKIN reconstructed human epidermal model. In the EpiSKIN test (Harlan 2014), duplicate tissues of reconstructed human skin were treated with 50 uL of the test substance during 3, 60 and 240 minutes. After treatment tissues were rinsed and incubated with MTT for 3 hours (at room temperature). Optical density of homogene solutions of MTT treated tissues was measured at 540 nm. As the test substance was shown to interfere with the MTT reduction, additional controls were included (non-viable tissue). The true viability was 74.7 -94.2% for the different exposure times, which is indicative for non-corrosive substances.

Eye irritation

In the SkinEthic assay (Harlan 2014) SkinEthic tissues were treated with 30 uL of the test substance in triplicate during 10 minutes. After treatment tissues (2 per treatment) were rinsed and treated with MTT. Optical density of extracted MTT solutions was measured at 540 nm. As the test substance interfered with MTT reduction to formazan, viability was expressed as percentage MTT conversion versus negative control with a correction for true viability (0.252). The true viability was 57.2%, which is indicative for irritant substances. As the assay is not formally validated, the results need to be regarded as preliminary, but worst case. In an in vivo study (Harlan 2015) the substance was installed in the eye of 2 rabbits (subsequently treated). The results are indicative for the classification as irritating to the eyes

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06-10-2012 to 09-10-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study performed according to the guideline and under GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Qualifier:
according to guideline
Guideline:
other: EU B40bis (EC440/2008)
GLP compliance:
yes (incl. QA statement)
Details on study design:
The EpiSKIN assay uses reconstructed human epidermis to assess skin corrosion. For the viability test enzymatic conversion of MTT to formazan is used.

In a pre-test direct interaction of the test substance with the detection chemical MTT is assessed. As the substance showed interference additional controls were added (water killed EPISKIN tissues).
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min
Value:
94.2
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min
Value:
74.7
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
240 min
Value:
84.4
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The test substance interfered with the reduction of MTT to blue formazan salt. Direct reduction compared to negative controls was 6.1 to 29.1% (validity criterion < 30%).

 

Exposure time

Mean corrected OD540

Correction applied

Relative mean viability

Negative control:

240 min

0.772

 

100 %

Positive controls:

240 min

0.029

 

3.8%

Test substance

3 min

60 min

240 min

0.727*

0.577*

0.655*

0.046#

0.197#

0.225#

94.2%

74.7%

84.8%

* TODTT = [ODTV – (ODkt - ODku)]

# correction value: ODkt - ODku

 

ODTV : chemical treated viable tissues

TODTT : true MTT metabolic conversion for treated tissue.

ODku : untreated killed tissues

ODkt: chemical treated killed tissues

 

 

Interpretation of results:
GHS criteria not met
Conclusions:
The test substance is non-corrosivein the EPISKIN reconstructed human epidermal model.
Executive summary:

In the EpiSKIN test, tissues of reconstructed human skin were treated with 50 uL of the test substance in duplicate during 3, 60 and 240 minutes. After treatment tissues were rinsed and incubated with MTT for 3 hours (at room temeprature). Thereafter tissues were allowed to dry, epidermis and collagen were separated, suspended (after vortexing) in isopropanol and kept overnight (all procedures in dark). Optical density of homogene solutions of MTT treated tissues was measured at 540 nm. As the test substance was shown to interfere with the MTT reduction, additional controls were included (non-viable tissue). Viability was expressed as the corrected percentage MTT conversion versus negative control. The true viability was 74.7 -94.2% for the different exposure times, which is indicative for non-corrosive substances.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06-02-2013 to 11-02-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study performed according to the guideline and under GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Principles of method if other than guideline:
exposure period 15 minutes with post-exposure incubation period of 42 hours
GLP compliance:
yes (incl. QA statement)
Details on study design:
The EpiSKIN assay uses reconstructed human epidermis to assess skin irritation. For the viability test enzymatic conversion of MTT to formazan is used.

In a pre-test direct interaction of the test substance with the detection chemical MTT is assessed. As the substance showed interference additional controls were added (water killed EPISKIN tissues).
In the main study triplicate tissues (both viable and killed) were treated with 30 uL of the test substance during 15 minutes. In addition triplicate tissues were treated with a negative control (DPBS) and a positive control (5% SDS) (non-viable tissues were treated with DPBS). After treatment tissues were rinsed, incubated in maintenance fluid for 42 hours at 37 °C and treated with MTT during 3 hours (at 37 °C). After MTT loading a total biopsy of each epidermis (epidermis and collagen) was made, that was suspended in acidified iso-propanol for 3 days and kept in the refrigerator at 1-10°C. Thereafter the optical density of the extracted MTT solutions was measured at 540 nm. Viability was expressed as percentage MTT conversion versus negative control with a correction for true viability according to EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals. ( TODTT = [ODTV – (ODkt - ODku)] )
Tissue viability > 50% means non-irritant
Tissue viability ≤ 50% means irritant
Irritation / corrosion parameter:
% tissue viability
Value:
109.2
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The test substance interfered with the reduction of MTT to blue formazan salt (correction factor 0.109).

 

Mean corrected OD540± SD

Correction applied

Relative mean viability ± SD

Negative control:

0.882 ± 0.056

 

100 ± 6.9%

Test substance:

0.898 ± 0.055*

0.109#

109.2 ± 6.7%

SDS:

0.064 ± 0.005

 

7.8 ± 0.6%

* TODTT = [ODTV – (ODkt - ODku)]

# correction value: ODkt - ODku

 

ODTV : chemical treated viable tissues

TODTT : true MTT metabolic conversion for treated tissue.

ODku : untreated killed tissues

ODkt: chemical treated killed tissues

 

Interpretation of results:
GHS criteria not met
Conclusions:
The test substance is non-irritant in the EPISKIN reconstructed human epidermal model.
Executive summary:

In this assay reconstructed human skin tissues were treated with 30 uL of the test substance in triplicate during 15 minutes. After treatment tissues were rinsed, kept for 42 hours and treated with MTT. Optical density of extracted MTT solutions was measured at 540 nm. As the test substance was shown to interfere with the MTT reduction, additional controls were included (non-viable tissue). Viability was expressed as the corrected percentage MTT conversion versus negative control. The viability was 109.2%, which is indicative for non-irritant substances.

The test substance is non-irritant in the EPISKIN reconstructed human epidermal model.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26-01-2015 to 09-03-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study according to the guidelines under GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Leicestershire, UK
- Strain: New Zealand White (Hsdlf:NZW)
- Age at study initiation: 12 and 20 weeks
- Weight at study initiation: 2.6 and 3.1 kg
- Housing: individually
- Water: ad libitum
- Feed: 2930C Teklad Global Rabbit diet ad libitum
- Acclimation period: ≥ 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23 °C
- Humidity (%): 30-70%
- Air changes (per hr): ≥ 15/hour
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
TEST MATERIAL: 0.1 mL
Observation period (in vivo):
Animal 1 21 days, Animal 2 14 days
Scoring at 1, 24, 46 and 72 h and 7, 14 and 21 days after instillation
Number of animals or in vitro replicates:
2
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): none

SCORING SYSTEM: Draize with calculations of irritation score according to modified version of the system described by Kay J.H. and Calandra J.C. (1962))

TOOL USED TO ASSESS SCORE: light of ophthalmoscope
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
fully reversible within: 21 days
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
fully reversible within: 14 days
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
2
Reversibility:
fully reversible within: 21 days
Irritation parameter:
conjunctivae score
Remarks:
discharge
Basis:
animal #1
Time point:
24/48/72 h
Score:
3
Max. score:
3
Reversibility:
fully reversible within: 21 days
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
3
Max. score:
3
Reversibility:
fully reversible within: 14 days
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #2
Time point:
24/48/72 h
Score:
2
Max. score:
2
Reversibility:
fully reversible within: 14 days
Irritation parameter:
conjunctivae score
Remarks:
discharge
Basis:
animal #2
Time point:
24/48/72 h
Score:
>= 1 - <= 2
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
2
Max. score:
2
Reversibility:
fully reversible within: 14 days
Other effects:
Body weight gain within normal ranges
No othetr signs of toxicity or pain

Animal no

1

2

1

24

48

72

7

14

21

1

24

48

72

7

14

cornea

1

1

1

1

1

1

0

0

0

0

0

0

0

iris

1

1

1

1

1

0

0

1

1

1

1

0

0

conjunctivae

 

 

 

 

 

 

 

 

 

 

 

 

 

redness

2

2

2

2

2

1

0

2

2

2

2

1

0

chemosis

3

3

3

3

1

0

0

2

2

2

2

1

0

discharge

3

3

3

3

2

1

0

3

2

1

1

0

0

 

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
The substance is irritating to the eyes
Executive summary:

The substance was installed in the eye of 2 rabbits (subsequently treated). The results are indicative for the classification as irritating to the eyes (category 2).

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12-03-2013 to 14-03-2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: not formally validated study method
Principles of method if other than guideline:
The SkinEthic reconstructed human corneal epithelium assay is used
GLP compliance:
yes (incl. QA statement)
Details on study design:
The SkinEthic reconstructed human corneal epithelium assay uses corneal epithelial tissue to assess penetration and concomittant cytotoxicity of the test substance.

In a pre-test direct interaction of the test substance with the detection chemical MTT is assessed. As the substance showed interference additional controls were added (freeze killed SkinEthic HCE tissues).
In the main study SkinEthic tissues were treated with 30 uL of the test substance during 10 minutes in triplicate. In addition a negative control (Na2HPO4 0.142 g/L; glucose 1.802 g/L; HEPES 7.149 g/L; KCl 0.224 g/L; NaCl 7.597 g/L) and a positive control (2% SDS) were included. After treatment tissues (2 per treatment) were rinsed and treated with MTT (incubation for 3 hours). Optical density of isopropanol extracted MTT solutions was measured at 540 nm. Viability was expressed as percentage MTT conversion versus negative control with a correction for true viability according to EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals. ( TODTT = [ODTV – (ODkt - ODku)] )
Tissue viability ≥ 60% means non-irritant
Tissue viability < 60% means irritant
The third tissue per treatment was preserved for histopathology.
Irritation parameter:
other: % viability
Value:
57.2
Negative controls validity:
valid
Positive controls validity:
valid
Other effects:
The test substance interfered with the reduction of MTT to blue formazan salt (correction factor 0.252).
The validity criteria for the viability of positive control, < 60%, was met.

Mean OD540

Correction applied

 

Relative mean viability

Negative control:

0.976*

 

100%

Test substance:

0.558*

0.252#

57.2%

SDS:

0.168*

 

17.2%

* TODTT = [ODTV – (ODkt - ODku)]

# correction value: ODkt - ODku

 

ODTV : chemical treated viable tissues

TODTT : true MTT metabolic conversion for treated tissue.

ODku : untreated killed tissues

ODkt: chemical treated killed tissues

No histopathology was performed.

Interpretation of results:
study cannot be used for classification
Conclusions:
The test substance is irritant in the SkinEthic reconstructed human corneal epithelium assay.
Executive summary:

In this assay SkinEthic tissues were treated with 30 uL of the test substance in triplicate during 10 minutes. After treatment tissues (2 per treatment) were rinsed and treated with MTT. Optical density of extracted MTT solutions was measured at 540 nm. As the test substance interfered with MTT reduction to formazan, viability was expressed as percentage MTT conversion versus negative control with a correction for true viability (0.252). The true viability was 57.2%, which is indicative for irritant substances. As the assay is not formally validated, the results need to be regarded as preliminary, but worst case.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The substance is non-corrosive and non-irritant in the in vitro assays. As these assays are sufficiently validated, no further testing is considered necessary and the substance is considered non-irritant.

In the non-validated in vitro assay for eye irritation a slightly positive result was obtained. This result was confirmed by an in vivo test.

Justification for classification or non-classification

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

The available data on eye irritation of the test substance are indicative for eye irritation and , in a worst case approach, it is concluded that the substance meets the criteria for classification as H319 (causes serious eye irritation Category 2) according to Regulation (EC) 1272/2008 .