Registration Dossier

Administrative data

Description of key information

Skin corrosion; OECD 431; Payne, R. (2017): Not classified

Skin irritation; OECD 439; Payne, R. (2017): Skin irritant category 2

Eye corrosion; OECD 437; Payne, R. (2017): Inconclusive

Eye irritation; OECD 492; Spohr, C. (2018): Substance has eye irritating potential

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 January - 04 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
Council Regulation (EC) No 440/2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Three-dimensional human skin model, comprising a reconstructed epidermis with a functional stratum corneum supplied by In Vitro Life Science Laboratories, Bratislava, Slovak Republic
Details on animal used as source of test system:
N/A
Justification for test system used:
Guideline specific test system.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: MatTek, In Vitro Life Science Laboratories, Bratislava, Slovak Republic
- Tissue batch number(s): 23397
- Production date: 01 Mar 2017
- Shipping date: 01 Mar 2017
- Delivery date: 01 Mar 2017
- Date of initiation of testing: 01 Mar 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ºC
- Temperature of post-treatment incubation (if applicable): 37 ºC

REMOVAL OF TEST MATERIAL AND CONTROLS - Volume and number of washing steps: the tissues were washed with phosphate buffered saline (PBS) to remove residual material. The rinsed tissues were kept in 24-well plates (holding plates) until all tissues were dosed and rinsed
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Not reported
- Wavelength: 570 nm
- Filter: Not reported
- Filter bandwidth: Not reported
- Linear OD range of spectrophotometer: Not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Pass - details provided in CoA (OD540-570 was between 1.0 and 3.0).
- Barrier function: Pass - details provided in CoA (ET-50 was between 4.77 and 8.72 h).
- Morphology: Not reported
- Contamination: Pass - details provided in CoA (no contamination - tested for HIV-1-virus; Hep B; Hep C; bacteria; yeast and other fungi).
- Reproducibility: Coefficient of Variation between tissue replicates was ≤ 30%.

NUMBER OF REPLICATE TISSUES: 4 per test group (2 each for 3 min and 60 min exposure)

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item did not reduce MTT.

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%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): Unchanged/ undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): N/A
- Concentration (if solution): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): Purified water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 8 M
Duration of treatment / exposure:
3 mins and 60 mins
Duration of post-treatment incubation (if applicable):
3 h MTT incubation followed by overnight isopropnaol extraction.
Number of replicates:
2 per treatment and exposure group
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min exposure (repeat)
Value:
89.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min exposure (repeat test)
Value:
52
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Not reported

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 1.357 for the 3-Minute exposureperiod and 1.338 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 6.6 and 8.0 % relative to the negative control following the 3 and 60-,minute exposure periods, respectively. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.
- Range of historical values if different from the ones specified in the test guideline: All values for the positive and negative control were within the historical ranges achieved by the testing facility in previous tests (Feb 2008 - Feb 2017, n=46), thus confirming the acceptable functioning of the test system.

Table 1      Relative mean viabilities for each treatment group

 

Exposure Period

Percentage Viability (% SD / % CoV)*

Negative Control§

Positive Control

Test Item

3 minutes

100 (0.068 / 6.0)

6.2 (0.078 / 20.6)

89.1 (0.090 / 8.8)

60 minutes

100 (0.061 / 4.9)

-1.4 (0.034 / 23.2)

52.0 (0.022 / 3.4)

*mean of 2 replicates

§negative controls set to 100 %

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be non-corrosive to the skin under the conditions of the test.
Executive summary:

OECD 431 (2017) - The skin corrosivity potential of Reaction Mass of N,N,N’,N’-tetrabutylmethylenediamine and dibutylamine was assessed using an EpiDerm Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD guidance 431 and GLP.

 

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 mins. At the end of the exposure period the test item was rinsed from each tissue before being loaded with MTT. After MTT loading each tissue was placed in 2 mL isopropanol for MTT extraction. After extraction, each tissue was pierced and the extraction solution aliquoted for absorbance measurements. Absorbency at 570 nm of each well was measured using a spectrophotometer.

The test item was considered to be non-corrsive to the skin under the conditions of the test.

 

Mean viability of tissues exposed to the test substance after 3 and 60 minutes were 89.1 and 52.0 %, respectively. The quality criteria required for acceptance of the results was met.

 

Under the conditions of this study the test substance is not considered to be corrosive to the skin.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 March - 21 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Commission Regulation (EC) No 761/2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 2-8 ºC, in darkness
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No, investigations of MTT and colour interference concluded that the test item did not interfere with the test system.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material): Applied as supplied

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): N/A

OTHER SPECIFICS: N/A
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDermTM SIT (EPI-200) three-dimensional human skin model, (lot 25800) comprising a reconstructed epidermis with a functional stratum corneum, supplied by MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
Source strain:
other: N/A
Details on animal used as source of test system:
N/A
Justification for test system used:
Guideline specific test system
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm SIT (EPI-200) 3D human skin model (supplied by MatTek In Vitro Life Science, Slovakia)
- Tissue batch number(s): 25803
- Production date: 05 April 2017
- Shipping date: 05 April 2017
- Delivery date: 05 April 2017
- Date of initiation of testing: 05 April 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 ºC
- Temperature of post-treatment incubation (if applicable): 37 ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsed with PBS (minimum of 3 rinses)
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: not reported
- Wavelength: 570 nm
- Filter: Not reported
- Filter bandwidth: Not reported
- Linear OD range of spectrophotometer: Not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: acceptable (provided in CoA; OD540-570 between 1.0-3.0)
- Barrier function: acceptable (provided in CoA; ET-50 between 4.77 and 8.72 h)
- Contamination: acceptable (provided in CoA; no contamination - screened for HIV-1-virus; Hep B and Hep C; bacteria, yeast and other fungi)

NUMBER OF REPLICATE TISSUES: 3 per test group

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- n/a

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after exposure is ≤ 50 %, realtive to the negative control.
- The test substance is considered to be non-irritant to skin if the viability after exposure is > 50 %, relative to the negative control.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: N/A, as per OECD 439 guidance
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
- Concentration (if solution): Undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): N/A
- Concentration (if solution): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): phosphate buffered saline (PBS) - concentration not reported

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5 % w/v
Duration of treatment / exposure:
60 mins exposure
Duration of post-treatment incubation (if applicable):
Post exposure and rinsing the tissues were placed on the appropriate medium and incubated for 41 hours and 20 minutes.

Upon completion of the recovery period, each tissue was rinsed with PBS before being placed on 0.3 mL of 1 mg/mL MTT in PBS and incubated for three hours at 37 ºC. Following MTT immersion, the samples were extracted in isopropanol for 2 hours.
Number of replicates:
3 per test group
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean of three test item replicates
Value:
3.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Not reported

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The OD values for the negative controls were between ≥0.8 and ≤2.8 (1.117).
- Acceptance criteria met for positive control: Viability of tissues was ≤ 20 % (4.9 %).
- Acceptance criteria met for variability between replicate measurements: Yes, ≤18 % (ca. 6 and 7 % for the negative and positive control groups, respectively).
- Range of historical values if different from the ones specified in the test guideline: Both negative and positive control values fall within the current historical control ranges.

Table 2      Mean OD570Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

 

Item

OD570of tissues

(corrected mean)

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

% COV Relative mean viability (%)

Negative Control Item

1.191

106.6

100

5.848

5.848

1.091

97.7

1.068

95.7

Positive Control Item

0.059

5.3

4.9

0.329

6.713

0.052

4.6

0.054

4.8

Test Item

0.038

3.4

3.5

0.557

15.907

0.034

3.0

0.046

4.1

OD= Optical Density

SD= Standard deviation

 

*= The mean viability of the negative control tissues is set at 100 %

 

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Under the conditions of this study the test substance is considered to be irritant to the skin and can be preliminarily classfied as Category 2 in accordance with UN GHS and EU CLP regulation.
Executive summary:

OECD 439 (2017) - The skin irritation potential of Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine was assessed using an EpiSkin Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD Guidance 439 and GLP.

 

Triplicate tissues were exposed to the test item for 60 minutes. At the end of the exposure period the test item was rinsed and the tissues incubated for a further 40 h in the presence of maintenance solution which would be used for possible inflammatory mediator determination. Each tissue was then loaded with MTT for 3 h and any resultant colour extracted. After incubation and extraction with isopropanol, the solutions were aliquoted for absorbance measurements. Absorbency at 570 nm of each well was measured using a spectrophotometer.

 

Mean viability of tissues exposed to the test substance after 60 minutes were 3.5 %. It was considered unnecessary to perform IL-1a analysis as the results of the MTT test were unequivocal. The quality criteria required for acceptance of the results was met.

 

Under the conditions of this study the test substance is considered to be irritant to the skin and can be preliminarily classfied as Category 2 in accordance with EU CLP regulation.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 March - 11 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.
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:
26 July 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:
Council Regulation (EC) No 440/2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity:
N/A
- Specific activity:
N/A
- Locations of the label:
N/A
- Expiration date of radiochemical substance:
N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Room temperature, in darkness
- Stability under test conditions:
Asumed stable
- Solubility and stability of the test substance in the solvent/vehicle:
N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:
No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
N/A
- Preliminary purification step (if any):
N/A
- Final dilution of a dissolved solid, stock liquid or gel:
N/A
- Final preparation of a solid:
N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Applied as supplied

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)
N/A

OTHER SPECIFICS: N/A
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Corneas from bovine eyes were obtained from a local abattoir
- Number of animals: 9 corneas were prepared
- Characteristics of donor animals (e.g. age, sex, weight): < 30 months old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were removed after slaughter, completely immersed in Hanks’ Balanced Salt Solution (containing penicillin at 100 IU/mL and streptomycin at 100 μg/mL) in a suitably sized container and transported on the same day to the testing facility.
- Time interval prior to initiating testing: Same day
- indication of any existing defects or lesions in ocular tissue samples: Only corneas free from such defects were used.
- Indication of any antibiotics used: During transport, eyes were immersed in Hanks’ Balanced Salt Solution (containing penicillin at 100 IU/mL and streptomycin at 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 ¨¨
- Concentration (if solution): Applied as supplied

VEHICLE
- Amount(s) applied (volume or weight with unit): N/A
- Concentration (if solution): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A
Duration of treatment / exposure:
10 mins
Duration of post- treatment incubation (in vitro):
After washing, the corneas were incubated (horizontally) for 2 hours and 2 minutes after which, corneal opacity was measured and then the anterior chamber emptied. For the permeability endpoint, 1 mL of sodium fluorescein (4 mg/mL solution in 0,9% sodium chloride) was added into the anterior chamber, while the posterior chamber was filled with fresh MEM, and the corneas were incubated in the vertical position for 1.5 hours at 32±1°C. Following this period, the media in the posterior chamber was removed, mixed and held in a labelled tube. Three 350 μL aliquots of this media (per cornea) were analysed for optical density at 490 nanometers (OD490) using a spectrophotometer.
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
:

On arrival at the test facility the eyes were carefully examined for defects including increased opacity, scratches and neovascularisation. Only corneas free from such defects were used.

QUALITY CHECK OF THE ISOLATED CORNEAS

Upon arrival at the test facility, the corneas were excised from the eyes and loaded onto the specifically designed holders that consist of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea. The holders were uniquely identified by a number written in indelible ink. Both chambers of each holder were filled with pre-warmed Minimal Essential Medium (MEM), with the posterior chamber filled first, ensuring that no bubbles were formed. The holders were incubated at 32±1°C for 1 hour. After the incubation, the media was removed from both the anterior and posterior chambers. Fresh media was added to the posterior chamber first and then the anterior chamber (this media replacement order ensured the cornea retained its natural curvature as much as possible). The opacity of each cornea was measured using an opacitometer. Any corneas found to have scratches or increased neovascularization or an opacity of >7 opacity units when examined prior to treatment were discarded.

NUMBER OF REPLICATES

3 per test group

NEGATIVE CONTROL USED

Yes, the negative control substance was 0.9% sodium chloride solution (batch number 15107BA1A, expiry date 06 April 2017), supplied by Baxter Healthcare Ltd., Thetford, UK.

SOLVENT CONTROL USED (if applicable)

N/A

POSITIVE CONTROL USED

Yes, the positive control substance was 100% dimethylformamide (DMF) (batch number SZBF1680V expiry date 26 October 2017), supplied by Sigma-Aldrich Company Ltd., Poole, UK.

APPLICATION DOSE AND EXPOSURE TIME

A volume of 750 μL of the test article was applied to each of three corneas followed by a 10 minute incubation at 32 °C.

TREATMENT METHOD:

Closed chamber

POST-INCUBATION PERIOD:

After opacity was measured, the corneas were incubated (horizontally) for 2 hours after which, corneal opacity was measured and then the anterior chamber emptied. For the permeability endpoint, 1 mL of sodium fluorescein (4 mg/mL solution in 0.9% sodium chloride) was added into the anterior chamber, while the posterior chamber was filled with fresh MEM, and the corneas were incubated in the vertical position for 1.5 hours at 32±1°C.

REMOVAL OF TEST SUBSTANCE

- Number of washing steps after exposure period:
Each cornea was washed with media containing phenol red (as a pH indicator, to identify and monitor the rinsing of acidic or alkaline materials) until this indicator showed no pH effect occurring, demonstrating that the test article had been removed successfully. The corneas were then washed once in media without phenol red and the opacities measured.

- POST-EXPOSURE INCUBATION:

After opacity was measured, the corneas were incubated (horizontally) for 2 hours after which, corneal opacity was measured and then the anterior chamber emptied. For the permeability endpoint, 1 mL of sodium fluorescein (4 mg/mL solution in 0.9% sodium chloride) was added into the anterior chamber, while the posterior chamber was filled with fresh MEM, and the corneas were incubated in the vertical position for 1.5 hours at 32±1°C. Following this period, the media in the posterior chamber was removed, mixed and held in a labelled tube. Three 350 μL aliquots of this media (per cornea) were analysed for optical density at 490 nanometers (OD490) using a spectrophotometer.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opaciometer used
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490)
- Others: Not conducted

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: As per OECD 437 guidance.
Irritation parameter:
in vitro irritation score
Run / experiment:
Test item mean IVIS
Value:
6.68
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Remarks:
No prediction can be made if the IVIS is >3 but ≤55
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No changes to the appearance of the corneas treated with the test article were noted.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Not reported.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Opacity and permeability values less than the established upper limits for background opacity and permeability values were reported.
- Acceptance criteria met for positive control: Positive controls IVIS fell within two standard deviations of the current historical mean (June 2009-May 2016, n=91)
- Range of historical values if different from the ones specified in the test guideline: Historical values reported.

Table 1      Corneal Opacity

 

Test Substance

Cornea Number

Initial Opacity

Post Incubation Opacity

Change in Opacity

Mean Change in Opacity

Corrected Opacity

Mean Corrected Opacity

Test article

8

4

8

4

N/A

4.3

6.3

9

3

12

9

9.3

10

4

9

5

5.3

Negative

2

4

4

0

-0.33

0.3

0.0

3

4

3

-1

-0.7

4

3

3

0

0.3

Positive

14

4

61

57

N/A

57.3

69.0

15

4

88

84

84.3

16

3

68

65

65.3

 

Table 2      Corneal Permeability

 

Test Substance

Cornea Number

Mean Blank OD490

OD490

Corrected OD490

Mean Corrected OD490

Final Corrected OD490

Mean Group Corrected OD490

Test article

8

N/A

0.059

0.059

N/A

0.052

0.023

9

0.014

0.014

0.007

10

0.017

0.017

0.010

Negative

2

0.000

0.012

0.012

0.007

0.004

0.000

3

0.004

0.004

-0.003

4

0.006

0.006

-0.002

Positive

14

N/A

0.395

0.395

N/A

0.388

0.755

15

1.052

1.052

1.044

16

0.840

0.840

0.833

 

Table 3      Calculated IVIS

 

Test Chemical

Mean Opacity

Mean Permeability

IVIS (Mean Opacity + (15 x Mean Permeability))

Test article

6.3

0.023

6.68

Negative control

0

0

0

Positive control

69

0.755

80.33

 

 

Interpretation of results:
study cannot be used for classification
Conclusions:
Under the conditions of this study Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine produced an IVIS score of 6.68 and it was concluded that no prediction can be made in respect of its potential to cause eye irritation.

The assay was considered valid as the assay acceptance criteria were met.
Executive summary:

OECD 437 (2017) - The Bovine Corneal Opacity and Permeabilty (BCOP) test was conducted using Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine in accordance with 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" (2013).

 

Undiluted test item was applied evenly to the surface of three corneas before being washed off with media solution after 10 minute test item contact time. A negative and positive control group, each containing 3 corneas, were also prepared.

 

Measurements for corneal opacity were made after 2 h incubation in the horizontal position with fresh media. Measurements for corneal permeability were made following 1 h and 30 mins incubation in the vertical position with sodium fluorescein.

 

Corneal opacity and corneal permeability media solutions were analysed by a spectrophotometer at 490 nanometers (OD490).

 

The mean corrected opacity reading and permeability readings for the test item were 6.3 and 0.023, resulting in anIn VitroIrritation Score (IVIS) of 6.68.

 

It was concluded that under the condition of this study the test item produced an IVIS score of 6.68 and therefore, no prediction could be made in respect of its potential to cause eye irritation.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.
Qualifier:
according to
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:
July 2015
Deviations:
no
Qualifier:
according to
Guideline:
other: MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™
Version / remarks:
29 June 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 2-8 ºC, in the dark
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: Applied as supplied
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No, test item was not an MTT reducer and did not interfere with colour changes.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Applied as supplied

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) : N/A

OTHER SPECIFICS: N/A
Species:
human
Details on test animals or tissues and environmental conditions:
EpiOcular™ kits and MTT-100 kits are purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) were cultured on specially prepared cell culture inserts (MILLICELL™, 10 mm ø).

EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt (29 August 2017) of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 6-well plates.

Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70% isopropanol- or ethanol-soaked tissue paper. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL fresh Assay Medium at 37 °C and the EpiOcular™ tissues were incubated at standard culture conditions overnight (about 18 hours).
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): 50 µL
- Concentration (if solution): Applied as supplied

VEHICLE
- Amount(s) applied (volume or weight with unit): N/A
- Concentration (if solution): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A
Duration of treatment / exposure:
30 minutes
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
2
Details on study design:
- Details of the test procedure used

Pre Test Assessments

The test items ability to directly reduce MTT was assessed. For this purpose approximately 50 µl of the test item were added to a 1 mL of a 1.0 mg/mL MTT solution (in DMEM) in a glass tube and the mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for three hours. A control (50 µL of deionised water in 1 mL of 1.0 mg/mL MTT solution) was performed concurrently. The result indicated that the test item did not have MTT reducing effects. An additional test with freeze-killed tissues did not have to be performed.

The photometric properties of the test item after contact with water and isopropanol were assesed. For this purpose each 50 µL of the test item were added to 1.0 ml of water and to 2 ml isopropanol in a glass tube. The water mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for at least one hour, the isopropanol mixture or for 2 to 3 hours at room temperature. The result indicated that the test item did not dye the water or isopropanol and would therefore not interfere with the photometric assessment of MTT during the main test.

Main Test

After the overnight incubation, the tissues were pre-wetted with 20 µL of Ca++Mg++free-DPBS. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH) for 30 minutes.

After the 30 minute Ca++Mg++free-DPBS pre-treatment, the test and control items were tested by applying 50 µL topically on the EpiOcular™ tissues. The tissues were incubated at standard culture conditions for 30 minutes.

At the end of the 30 minutes treatment time, the test item was removed by extensively rinsing the tissues with Ca++Mg++-free DPBS (brought to room temperature). Three clean beakers containing a minimum of 100 mL each of Ca++Mg++-free DPBS were used per test item. Each test item utilized a different set of three beakers. The inserts containing the tissues were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control items were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The culture was then rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent material (to break the surface tension).

After rinsing, the tissues were immediately transferred to and immersed in 5 ml of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for 12 minute immersion incubation (post-soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue.

At the end of the post-soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the inserts were blotted on absorbent material, and transferred to the appropriate well of the pre-labelled glass tube containing 1 ml of warm Assay Medium. The tissues are incubated for 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).

After post-treatment incubation of 120 minutes the MTT assay was performed.

At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 ml of MTT solution. Once all the tissues are placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
Each insert was removed from the 24-well plate after 180 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plates were sealed with a standard plate sealer, and were stored overnight at 2-8 °C in the dark and then shaken for 2-3 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken.
The extract solution was mixed and two 200 µL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.

The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro, version 4.7.1). No reference wavelength measurement was used.

- RhCE tissue construct used, including batch number: EpiOcular kit (Lot: 23574). Supplied by MatTek Coorp., Slovakia.

- Doses of test chemical and control substances used: 50 µL applied topically.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): Tissues exposed for 30 minutes at 37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH. Tissues were immersed for 2 seconds during the rinsing process. Post exposue incubation was 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2.

- Justification for the use of a different negative control than ultrapure H2O (if applicable): N/A

- Justification for the use of a different positive control than neat methyl acetate (if applicable): N/A

- Description of any modifications to the test procedure: N/A

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): N/A

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): 2 tissues per test group (test item, positive and negative control). 1 blank tissue prepared with no treatment.

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm

- Description of the method used to quantify MTT formazan: See above.

- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable): N/A

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: The percent viability of each of the two relating tissues for each control and test item relative to the negative control (100% control) were calculated. The difference of the viability between duplicate tissues was calculated. If the difference is >20% the test is considered as non-qualified. The mean test item viability (TI viability) was calculated and the test item was classified according to the prediction model. If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labelled non-irritant. If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labelled irritant.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Yes

- Complete supporting information for the specific RhCE tissue construct used: Yes

- Reference to historical data of the RhCE tissue construct: Yes

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: Yes

- Positive and negative control means and acceptance ranges based on historical data: Yes
Irritation parameter:
other: Mean relative viability (%)
Run / experiment:
Test item mean value
Value:
31.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation

Table 1       Results after exposure to Results after treatment for 30 minutes with Reaction mass of N,N,N’,N’,tetrabutylmethylenediamine and dibutylamine and the controlsfor 30 minutes

 

Dose Group

AbsorbanceWell 1

(Tissue 1/2)

AbsorbanceWell 2 (Tissue 1/2)

Mean Absorbance* (Tissue 1/2)

Mean Absorbance* (Tissue 1 and 2)

Mean Absorbance of2 Tissues*

Rel. Absorbance [%]Tissue 1 and 2**

Absolute Value of the Difference of the Rel. Absorbances [%]Tissue 1 and 2

Mean Rel. Absorbance

[% of Negative Control]**

Blank

0.038

0.038

0.038

-

 

Negative Control

1.674

1.753

1.714

1.676

1.691

99.1

1.8

100.0

1.735

1.753

1.744

1.706

100.9

Positive Control

0.626

0.693

0.660

0.622

0.617

36.8

0.6

36.5

0.647

0.652

0.649

0.611

36.2

Test Item

0.616

0.663

0.640

0.602

0.539

35.6

7.4

31.9

0.517

0.512

0.515

0.477

28.2

* mean of 2 replicates after blank correction

** relative absorbance = ((100 • test item absorbance) / mean absorbance of the negative control)

 

Interpretation of results:
study cannot be used for classification
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, Reaction mass of N,N,N’,N’,tetrabutylmethylenediamine and dibutylamine possesses an eye irritating potential.
Executive summary:

OECD 492 (2018) - This in vitro study was performed to assess the eye irritation potential of Reaction mass of N,N,N’,N’,tetrabutylmethylenediamine and dibutylamine by means of the Human Cornea Model Test.

 

Additional tests with viable or freeze-killed tissues were not performed, since the colourless test item did not dye water or isopropanol, and did not prove to be a MTT reducer.

 

Tissues of the human cornea model EpiOcularwere treated with the test item, the positive and the negative control for 30 minutes each in duplicate.

 

Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 36.5 %, thus the validity of the test system is ensured.

 

Relevant irritating effects were observed following 30 minutes incubation with the test item. The mean relative absorption value of the tissues corresponding to the cornea viability decreased to 31.9 % compared with the value of the negative control (threshold for irritancy: ≤ 60%).

 

In conclusion, it can be stated that in this study and under the experimental conditions reported, Reaction mass of N,N,N’,N’,tetrabutylmethylenediamine and dibutylamine possesses an eye irritating potential.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation / Corrosion

OECD 431 (2017) - The skin corrosivity potential of Reaction Mass of N,N,N’,N’-tetrabutylmethylenediamine and dibutylamine was assessed using an EpiDerm Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD guidance 431 and GLP.  Duplicate tissues were treated with the test item for exposure periods of 3 and 60 mins. At the end of the exposure period the test item was rinsed from each tissue before being loaded with MTT. After MTT loading each tissue was placed in 2 mL isopropanol for MTT extraction. After extraction, each tissue was pierced and the extraction solution aliquoted for absorbance measurements. Absorbency at 570 nm of each well was measured using a spectrophotometer.  Mean viability of tissues exposed to the test substance after 3 and 60 minutes were 89.1 and 52.0 %, respectively. The quality criteria required for acceptance of the results was met.  Under the conditions of this study the test substance is not considered to be corrosive to the skin.

OECD 439 (2017) - The skin irritation potential of Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine was assessed using an EpiSkin Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD Guidance 439 and GLP.  Triplicate tissues were exposed to the test item for 60 minutes. At the end of the exposure period the test item was rinsed and the tissues incubated for a further 40 h in the presence of maintenance solution which would be used for possible inflammatory mediator determination. Each tissue was then loaded with MTT for 3 h and any resultant colour extracted. After incubation and extraction with isopropanol, the solutions were aliquoted for absorbance measurements. Absorbency at 570 nm of each well was measured using a spectrophotometer.  Mean viability of tissues exposed to the test substance after 60 minutes were 3.5 %. It was considered unnecessary to perform IL-1a analysis as the results of the MTT test were unequivocal. The quality criteria required for acceptance of the results was met.  Under the conditions of this study the test substance is considered to be irritant to the skin and can be preliminarily classified as Category 2 in accordance with EU CLP regulation.

Eye Irritation

OECD 437 (2017) - The Bovine Corneal Opacity and Permeabilty (BCOP) test was conducted using Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine in accordance with 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" (2013).  Undiluted test item was applied evenly to the surface of three corneas before being washed off with media solution after 10 minute test item contact time. A negative and positive control group, each containing 3 corneas, were also prepared.  Measurements for corneal opacity were made after 2 h incubation in the horizontal position with fresh media. Measurements for corneal permeability were made following 1 h and 30 mins incubation in the vertical position with sodium fluorescein.  Corneal opacity and corneal permeability media solutions were analysed by a spectrophotometer at 490 nanometers (OD490).  

The mean corrected opacity reading and permeability readings for the test item were 6.3 and 0.023, resulting in an In Vitro Irritation Score (IVIS) of 6.68.  It was concluded that under the condition of this study the test item produced an IVIS score of 6.68 and therefore, no prediction could be made in respect of its potential to cause eye irritation.

OECD 492 (2018) - This in vitro study was performed to assess the eye irritation potential of Reaction mass of N,N,N’,N’,tetrabutylmethylenediamine and dibutylamine by means of the Human Cornea Model Test. Additional tests with viable or freeze-killed tissues were not performed, since the colourless test item did not dye water or isopropanol, and did not prove to be a MTT reducer. Tissues of the human cornea model EpiOcular™were treated with the test item, the positive and the negative control for 30 minutes each in duplicate. Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 36.5 %, thus the validity of the test system is ensured. Relevant irritating effects were observed following 30 minutes incubation with the test item. The mean relative absorption value of the tissues corresponding to the cornea viability decreased to 31.9 % compared with the value of the negative control (threshold for irritancy: ≤ 60%). In conclusion, it can be stated that in this study and under the experimental conditions reported, Reaction mass of N,N,N’,N’,tetrabutylmethylenediamine and dibutylamine possesses an eye irritating potential.

Whilst the OECD 437 (BCOP) and OECD 492 (EpiOcular) methodology for eye irritation does not permit discrimination between serious eye damage and eye irritation in accordance with CLP, a conclusion on classification and labelling is reached for this substance based on a weight-of-evidence approach, based on the integrated testing approach conducted in accordance with ECHA Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7a.(v6.0 - July 2017). Both assays conducted indicate with certainty that the substance should not be classified for serious eye damage (Eye Dam. 1), yet both assays confirm that the substance should not have no classification with respect to eye irritation and is considered to have eye irritation potential. Therefore, the proposed classification and labelling scheme for this substance under CLP is category 2 (H319: causes serious eye irritation).

Justification for classification or non-classification

Reaction mass of N,N,N',N,-tetrabutylmethylenediamine and dibutylamine meets the classification for skin irritation (category 2) and eye irritation (category 2) according to Regulation (EC) No 1272/2008 (CLP).