(butylamine)[[2,2'-thiobis[4-(1,1,3,3-tetramethylbutyl)phenolato]](2-)-O,O',S]nickel

Endpoint summary

Description of key information

Skin irritation/corrosion: Not corrosive (OECD 431, GLP)

Skin irritation/corrosion: Not irritating (OECD 439, GLP)

Serious eye damage/eye irritation: Not irritating (OECD 437, GLP)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23/02/2017-02/03/2017

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Baoding 20161221
- Expiration date of the batch:20-12-2018
- Purity: 99.1%
- Purity test date: 21-12-2016

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 mins at room temperature and 60 minutes at 37±1°C
- Temperature of post-treatment incubation (if applicable): 37±1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: Tissues were thoroughly rinsed and blotted to remove the test substance/controls.
- Observable damage in the tissue due to washing: None noted
- Modifications to validated SOP: None

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Libra S22
- Wavelength: 570 nm

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL H2O

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): 8N KOH

Duration of treatment / exposure:

3 and 60 mins

Duration of post-treatment incubation (if applicable):

After rinsing, tissues were transferred to 24-well plates containing MTT medium and incubated at culture conditions for 3 hours.

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 mins

Value:

107

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

8.5

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes

Value:

93.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

3.9

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: The test substance did not change colour of MTT medium (see Figure 1). The test substance does not reduce MTT directly.

- Colour interference with MTT:
Water: The test substance is hydrophobic. After incubation water remained clear and colourless (see Figure 2).
Isopropyl alkohol : The test substance is insoluble. After incubation, the test substance solvent remained colourless, slightly clouded. The colour of the test substance did not interfere with evaluation.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The assay meets the acceptance criterion - OD570 of the NC tissues was 1.655 (3 min) and 1.709 (60 min) which is ≥ 0.8 and ≤ 2.8.

- Acceptance criteria met for positive control: Viability of tissues treated with 8N KOH after 60 minutes treatment was 3.9 % which is ＜15%

- Acceptance criteria met for variability between replicate measurements: CV values in all triplets of tissues were ≤ 0.3 (see Table 1).

Interpretation of results:

GHS criteria not met

Conclusions:

Under the above-described experimental design, the test substance UV-1084 was non-corrosive in In vitro Skin Corrosion Test on EpiDermTM tissues.

Executive summary:

In an in vitro skin corrosion assay in a human epidermal model EpiDerm (17-164), water-moistened reconstructed human epidermis (RhE) tissue was exposed to 25 mg of UV-1084 (99.1%) for 3 and 60 minutes. Sterile water was used for the negative control and 8N KOH was used for the positive control. After rinsing, tissues were incubated with MTT for 3 hours and then extracted with isopropyl alcohol for 2 hours at room temperature with shaking. The OD570 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

The colour of the test substance did not interfere with the endpoint. The test substance is not directly MTT reducing. The negative control gave the appropriate response. The average viability of tissues treated with the test substance UV-1084 was 107 % of the negative control average value after 3 minutes treatment and 93.4 % after 60 minutes treatment. The average viability of positive control tissues at 3 and 60 minutes was 8.5% and 3.9%, respectively. The test substance UV1084 was non-corrosive in the EpiDermTM model.

This in vitro skin corrosion study in the human epidermal model EpiDermTM is acceptable and satisfies the guideline requirement for an OECD 431 study.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10/05/2017 to 13/05/2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Baoding 20161221
- Expiration date of the batch:20-12-2018
- Purity: 99.1%
- Purity test date: 21-12-2016

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 25 minutes at room temperature and the remaining 35 minutes at 37±1°C
- Temperature of post-treatment incubation (if applicable): 37±1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: Tissues are then thoroughly rinsed with PBS
- Modifications to validated SOP: None

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer:Libra S22
- Wavelength:570 nm
- Filter: No external filter was used

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg of substance

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL
- Concentration (if solution):5 % SDS (in H2O)

Duration of treatment / exposure:

60±1 minutes

Duration of post-treatment incubation (if applicable):

After 24 hours and 20 minutes of post-incubation period, the medium was replaced by fresh one. Tissues were incubated for another 19 hours, 7 minutes.

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 mins

Value:

97.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

4

Other effects / acceptance of results:

- OTHER EFFECTS:
Direct MTT Reduction and Colour Interference
These tests were not performed as a part of this study. In the previously performed study (Study No. 30/17/4AC: butylamine)[[2,2’-thio-bis[4-(1,1,3,3-tetramethylbutyl)phenolato]](2-)-O,O’,S]nickel - In vitro Skin Corrosion Test (EpiDermTM Model); VUOS-CETA Report No. 17-164, 2017), direct MTT reduction with the test substance and colour interference was excluded.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 of the NC tissue was 1.682 which meets the acceptance criteria of ≥ 0.8 and ≤ 2.8. OD570 historical negative control range is 1.455-2.347 (42 cases).

- Acceptance criteria met for positive control: The mean viability of the PC tissues expressed as % of the negative control tissues is 4% which meets the acceptance criterion of ≤ 20 %. The 95±% confidence interval of historical positive control is 0-0.190 (42 cases).

- Acceptance criteria met for variability between replicate measurements:
The SD calculated from individual % tissue viabilities of the 3 identically treated replicates was 0.1 % for the positive control, 4.9 % for negative control and 8.2 % for the test substance what is < 18 % in all cases.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the above-described experimental design, the average viability of tissues treated by the test substance, UV-1084, was 97.3 % of negative control average value. UV-1084 was negative in the EpiDermTM model.

Executive summary:

In an in vitro skin irritation assay in a human epidermal model EpiDermTM (17-402), reconstructed human epidermis (RhE) tissue was exposed to 25 mg of UV-1084 (99.1%) for 60±1 minutes (25 minutes at room temperature and the remaining 35 minutes at 37±1°C). PBS was used for the negative control and 5% SDS was used for the positive control. After removal of the test substance, tissues were post-incubated for approximately 42 hours. Three hours incubation with MTT and a 2 hour isopropyl alcohol extraction period followed. The OD570 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

The colour of the test substance did not interfere with the endpoint. The test substance is not directly MTT reducing. The average viability of tissues treated by the test substance UV-1084 was 97.3 % of negative control average value i.e. viability was > 50 %. The average viability of tissues treated by the positive control (5% SDS) was 4 % of the negative control average value. According to these results, the test substance is not irritating.

This in vitro skin irritation study in the human epidermal model EpiDermTM is acceptable and satisfies the guideline requirement for an OECD 439 study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21.06.2017 – 22. 06. 2017

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Baoding 20161221
- Expiration date of the batch:20-12-2018
- Purity: 99.1%
- Purity test date: 21-12-2016

Species:

cattle

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source:Breeding service CHOVSERVIS a.s., division TORO® Hlavečník, Hradec Králové, Czech Republic

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
The test substance was tested neat by direct application onto the corneal surface.

Duration of treatment / exposure:

4 hours

Number of animals or in vitro replicates:

3 per group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS:
Eyes were collected by slaughterhouse employees. The eyes were enucleated as soon as possible after death. No detergent was used. Only healthy animals (12 to 30 months old) considered suitable for entry into the human food chain were used as a source of corneas for use in the BCOP test. The risk of contamination was minimized (e.g., by keeping the container containing the eyes on ice, by adding antibiotics to the HBSS used to store the eyes during transport (e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL). The time interval between collection of the eyes and use of corneas in the BCOP was minimized (the corneas were collected and used on the same day). The results were based on the selection criteria for the eyes, as well as the positive and negative control responses. All eyes used in the assay were from the same group of eyes collected on a specific day.

Corneas free of defects were dissected with a 2 to 3 mm rim of sclera remaining to assist in subsequent handling, with care taken to avoid damage to the corneal epithelium. Isolated corneas were mounted in specially designed corneal holders that consisted of anterior and posterior compartments, which interfaced with the epithelial and endothelial sides of the cornea, respectively. Both chambers were filled to excess with pre-warmed Eagle's Minimum Essential Medium (EMEM). The device was then equilibrated at 32 ± 1°C for at least one hour in water bath to allow the corneas to equilibrate with the medium and to achieve normal metabolic activity, to the extent possible. Following the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Any corneas that showed macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded. Each test group (test substance, concurrent negative and positive controls) consisted of the three eyes. The three corneas with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment and positive control groups.

QUALITY CHECK OF THE ISOLATED CORNEAS:
The eyes, once they arrive at the laboratory, were carefully examined for defects including scratched, and neovascularisation. Only corneas from eyes free of such defects were used. The isolated corneas, after achieve normal metabolic activity (inductive incubation at 32 ± 1°C for one hour), were examined again. The corneas that show macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or a baseline opacity >7 opacity units were discarded. From 20 eyes, one eye was eliminated after inductive incubation, because the cornea was damaged during installation of the cornea to the holder. Nine corneas were used for the study (the corneas No. 1, 2, 3, 4, 5, 6, 7, 8 and 9), 7 eyes were superfluous and the remaining 3 were used for the testing of another substance.


NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: 0.9% NaCl

POSITIVE CONTROL USED: 20% Imidazole in 0.9% NaCl

APPLICATION DOSE AND EXPOSURE TIME: The test substance was tested neat by direct application onto the corneal surface for 4 hours.

TREATMENT METHOD: The Open-chamber method was used, because the test substance could not be prepared as a suspension or solution in 0.9% NaCl or water. The test substance (enough to completely cover the cornea) was applied directly to the epithelial surface of the cornea using the spatula. After dosing, the glass window was replaced on the anterior chamber to recreate a closed system.


POST-INCUBATION PERIOD: No, substance is a solid.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Once with EMEM (containing phenol red) and once with EMEM alone.

- POST-EXPOSURE INCUBATION: The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity (as value of illuminance) was measured quantitatively with the aid of an opacitometer (Opacitometer, Kit OP3,0 – Duratec, Analysertechnik – Germany).
- Corneal permeability: 1 mL sodium fluorescein solution (5 mg/mL) was added to the anterior chamber of the corneal holder, which interfaced with the epithelial side of the cornea, while the posterior chamber, which interfaced with the endothelial side of the cornea, is filled with fresh EMEM. The holder was incubated in horizontal position for 1.5 hours at 32 ± 1 ºC.

The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured with the aid of UV/VIS spectrophotometry (Spectrophotometer GENESYSTM 10 UV/VIS Scanning). The values of absorbance measured at 490 nm were recorded as optical density (OD490) values. This term was used because the measuring is performed with visible light spectrophotometer using a standard 1 cm path length.

SCORING SYSTEM: IVIS = mean opacity value + (15 x mean permeability OD490 value)

DECISION CRITERIA: A test is considered acceptable if the positive control gives IVIS that falls within one standard deviations of the current historical mean, which is to be updated at least every three months, or each time an acceptable test is conducted in laboratories where tests are conducted infrequently. The negative or solvent/vehicle control responses should result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control.

Irritation parameter:

cornea opacity score

Value:

2.35

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

52.20

Irritation parameter:

fluorescein leakage

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

1.893

Irritation parameter:

in vitro irritation score

Value:

2.35

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

2.5

Positive controls validity:

valid

Remarks:

80.6

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Appearance of corneas was observed before and after application of the test substance, negative and positive control (see Tables 2 and 3). No macroscopic damage was observed on corneas before application. Corneal opacity was observed on the corneas treated by positive control. The corneas treated by negative control were without macroscopic damage. The corneas treated by the test substance were without macroscopic damage.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The value of opacity for the negative control (0.9% NaCl) obtained during the study was 2.03 and the value of permeability was 0.031. The values obtained during this study did not exceed the upper limits of the historical controls (2.68 ±0.92 and 0.0295 ±0.0127) from 3* studies, so the study is considered acceptable.

*The negative historical control only refers to 3 studies as a new opacitometrer was purchased in Feb.2017


- Acceptance criteria met for positive control: The value of IVIS for positive control (20% imidazole) obtained during the study was 80.6. This value is within the acceptance limit (one standard deviations (4.55) of the current historical mean (77.39) of 3* studies), so the study is considered acceptable.

*The positive historical control only refers to 3 studies as a new opacitometrer was purchased in Feb.2017

Decision criteria:

The IVIS cut-off value for identifying the test substance as including serious eye damage (UN GHS Category 1) and the test substance not requiring classification for eye irritation or serious damage (UN GHS No Category) will be given hereafter:

 

Table 1:Decision criteria

IVIS	UN GHS
≤ 3	No Category
>3; ≤ 55	No prediction can be made
> 55	Category 1

UN GHS Category 1: “Serious eye damage”

UN GHS Category 2: “Eye irritation”

UN GHS No Category: Chemicals that do not meet the requirements for classification as: UN

GHS Category 1 or 2. Interchangeable with “Not Classified”

Interpretation of results:

GHS criteria not met

Conclusions:

In the Bovine Corneal Opacity and Permeability (BCOP) assay, the IVIS for UV-1084 was 0.36. As the IVIS is ≤ 3, the classification of the test substance according to UN GHS criteria for eye irritation or serious eye damage is: No category.

Executive summary:

In the Bovine Corneal Opacity and Permeability (BCOP) assay (17-450), isolated bovine corneas were exposed to neat UV-1084 for 4 hours using the open chamber method. 0.9% NaCl was used for the negative control and 20% Imidazole in 0.9% NaCl was used for the positive control. The corneas were rinsed twice and the opacity and permeability (via sodium fluorescein dye) of each cornea were recorded.

There was no macroscopic damage to any corneas treated with the test substance. The mean opacity value and mean permeability OD490 for the test substance were 2.35 and 0.0 respectively. The IVIS for the test substance was 2.35. The positive control gave the appropriate response. The IVIS for UV-1084 is ≤ 3 and the classification of the test substance according to UN GHS criteria for eye irritation or serious eye damage is: No category.

This Bovine Corneal Opacity and Permeability (BCOP) is acceptable and satisfies the guideline requirement for an OECD 437 study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion

There is one in vitro skin corrosion study and one in vitro skin irritation study available.

In an in vitro skin corrosion assay in a human epidermal model EpiDerm (OECD 431/GLP), water-moistened reconstructed human epidermis (RhE) tissue was exposed to 25 mg of UV-1084 (99.1%) for 3 and 60 minutes. Sterile water was used for the negative control and 8N KOH was used for the positive control. After rinsing, tissues were incubated with MTT for 3 hours and then extracted with isopropyl alcohol for 2 hours at room temperature with shaking. The OD570 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues. The colour of the test substance did not interfere with the endpoint. The test substance is not directly MTT reducing. The negative control gave the appropriate response. The average viability of tissues treated with the test substance UV-1084 was 107 % of the negative control average value after 3 minutes treatment and 93.4 % after 60 minutes treatment. The average viability of positive control tissues at 3 and 60 minutes was 8.5% and 3.9%, respectively. The test substance UV1084 was non-corrosive in the EpiDermTM model.

In an in vitro skin irritation assay in a human epidermal model EpiDermTM (OECD 439/GLP), reconstructed human epidermis (RhE) tissue was exposed to 25 mg of UV-1084 (99.1%) for 60±1 minutes (25 minutes at room temperature and the remaining 35 minutes at 37±1°C). PBS was used for the negative control and 5% SDS was used for the positive control. After removal of the test substance, tissues were post-incubated for approximately 42 hours. Three hours incubation with MTT and a 2 hour isopropyl alcohol extraction period followed. The OD570 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues. The colour of the test substance did not interfere with the endpoint. The test substance is not directly MTT reducing. The average viability of tissues treated by the test substance UV-1084 was 97.3 % of negative control average value i.e. viability was > 50 %. The average viability of tissues treated by the positive control (5% SDS) was 4 % of the negative control average value. According to these results, the test substance is not irritating.

Serious eye damage/eye irritation

There is one in vitro eye damage/irritation study available.

In the Bovine Corneal Opacity and Permeability (BCOP) assay (OECD 437/GLP), isolated bovine corneas were exposed to neat UV-1084 for 4 hours using the open chamber method. 0.9% NaCl was used for the negative control and 20% Imidazole in 0.9% NaCl was used for the positive control. The corneas were rinsed twice and the opacity and permeability (via sodium fluorescein dye) of each cornea were recorded. There was no macroscopic damage to any corneas treated with the test substance. The mean opacity value and mean permeability OD490 for the test substance were 2.35 and 0.0 respectively. The IVIS for the test substance was 2.35. The positive control gave the appropriate response. The IVIS for UV-1084 is ≤ 3 and the classification of the test substance according to UN GHS criteria for eye irritation or serious eye damage is: No category.

The results from these studies are acceptable to use in the human health hazard assessment.

Justification for classification or non-classification

Based on the available information in the dossier, the substance UV-1084 (CAS No. 14516-71-3) does not need to classified  for skin irritation/corrosion and does not need to classified for serious eye damage/eye irritation when considering the criteria outlined in Annex I of 1272/2008/EC.