Copper, diammine[5,5'-bi-1H-tetrazolato(2-)-kN1, kN1']

Endpoint summary

Description of key information

According to in vitro skin irritation (reconstructed human epidermis test method, OECD 439) and in vitro skin corrosion (reconstructed human epidermis test method, OECD 431) assays, CuDABT is not classified as irritant or corrosive for the skin.

According to in vitro eye irritation assay (Bovine corneal opacity and permeability test method, OECD 437), CuDABT is not classified as irritant or corrosive for the eyes.

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:

June-July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

dated 28 july 2015
method B40bis of the Council regulation No.440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Provider : AUTOLIV ASP, 16700 W. Hwy 83, Promontory, UTAH 84307, USA
- batch No. P3656461
- Expiration date of the lot/batch: august 25th 2020
- Purity: 97.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in a restricted area dedicated to explosive products, at outside temperature and humidity
- Solubility and stability of the test substance in the solvent/vehicle: not soluble in water

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no treatment

FORM AS APPLIED IN THE TEST (if different from that of starting material)
not different

Test system:

artificial membrane barrier model

Source species:

human

Cell type:

other: 0.6 cm2 reconstituted epidermis

Cell source:

other: epiCS, Cell Systems

Justification for test system used:

corrosion assay on animals is forbiden

Vehicle:

unchanged (no vehicle)

Details on test system:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): no
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 20 times

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Elx80 absorbance microplate reader Biotek
- Wavelength:570 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if viability measured is <50% after 3 min of exposure or if viability measured is >=50% after 3 min and <15% after 60 minutes of exposure
- The test substance is considered to be non-corrosive to skin if viability measured is >= 50% after 3 min and >=15% after 60 minutes of exposure

Control samples:

yes, concurrent negative control

yes, concurrent no treatment

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied 25 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): PBS PAN BIOTECH GmbH, batch No. 23802016


POSITIVE CONTROL
- Amount(s) applied (volume or weight): 8N KOH, Sigma, batch No. SLBD3295V

Duration of treatment / exposure:

3 minutes
30 minutes

Duration of post-treatment incubation (if applicable):

no applicable

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

test item: after 3 minutes: 95.11% of viability after 60 minutes: 94.25% of viability

Value:

> <

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:
- Direct-MTT reduction: the intrinsic colour of the test item is blue, wich did not allow to conclude on its direct >MTT reduction potential. 2 killed skin models were added.
- Colour interference with MTT: not interfere with MTT assay

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
- Acceptance criteria met for positive control:
- Acceptance criteria met for variability between replicate measurements:
- Range of historical values if different from the ones specified in the test guideline:

Interpretation of results:

GHS criteria not met

Conclusions:

In accordance with the regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude taht the test item Copper Dianmmine Bitetrazole (CuDABT) does not have to be classified in category 1 "corrosive". The hasard statement "H314: causes severe skin burns and eye damage" with the signal word "Danger" are not required.

Executive summary:

The aim of the study was to evaluate the possible corrosive effects of the test item after topical administration on in vitro human reconstituted epidermis (epiCS(R), CellSystems(R)).

The test item Copper Diammine Bitetrazole (CuDABT) was applied as supplied, at the dose of 25 mg, to 2 living and 2 killed Human skin model surfaces (0.6 cm2, epiCS(R), CellSystems(R)) during 3 minutes and 1 hour, followed by a rince with 20 mL of PBS. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The experimental protocol was established in accordance with O.E.C.D. test Guideline No. 431 dated 28 July 2015 and the method B.40bis of the Council regulation No.440/2008.

3 minutes and 1 hour after the test item application, the mean corrected percents viability of epidermis skins treated with the test item were 95.11% and 94.25%, versus 5.13% and 0.39%, respectively, with the positive control item (potassium hydroxide 8N).

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item Copper Diammine Bitetrazole (CuDABT) does not have to be classified in Category 1 'Corrosive". The hazard statement "H314: Causes severe skin burns and eye damage" with signal word "Danger" are not required.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 23th to May 20th 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

study performed in accordance with guideline OECD439 and GLP. A minor deviation about teperature of incubation (bteween 37.0 to 38.2°C instead of 37°C +/- 1°C) but conidered have no significant impact on the result and the validity of the study.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 28 July 2015

Deviations:

yes

Remarks:

incubation temperature deviation, considered without impact on results of the study.

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

Council regulation No. 761/2009 dated 23 July 2009

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Provider : AUTOLIV ASP, 16700 W. Hwy 83, Promontory, UTAH 84307, USA
- batch No. P3656461
- Expiration date of the lot/batch: august 25th 2020
- Purity: 97.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in a restricted area dedicated to explosive products, at outside temperature and humidity
- Solubility and stability of the test substance in the solvent/vehicle: not soluble in water

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no treatment

FORM AS APPLIED IN THE TEST (if different from that of starting material)
not different

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Vehicle:

unchanged (no vehicle)

Details on test system:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C, 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 25 x 1mL
- Observable damage in the tissue due to washing: blue spots observed


DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT 1.0 mg/mL, 300µL per 0.5 cm2 reconstructed epidermis, 2h55 incubation at 37°C, 5%CO2
- Spectrophotometer: ELx800 absorbance microplate reader, BioTek
- Wavelength: 570 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-exposure incubation is <=50%
- The test substance is considered to be non-irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-exposure incubation is > 50%

Control samples:

yes, concurrent negative control

yes, concurrent no treatment

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 0.5 g of SDS (SIGMA batch No. STBF1623V) in 10 mL of distilled water
- Concentration (if solution): 5% SDS

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean corrected percent viability of the treated tissues was 82.1%

Value:

> 0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

In accordance with the Regulation (EC) No. 1272/2008, the test item Copper Diammine Bitetrazole (CuDABT) has to be considered as Non-irritant to skin in accordance with UN GHS No Category. No hazard statement or signal word is required

Executive summary:

The aim of the study was to evaluate the possible irritating effects of the test item after topical administration on in vitro human reconstituted epidermis (SkinEthic RHE model).

The test item Copper Diammine Bitetrazole (CuDABT) was applied as supplied, at the dose of 16 mg, to 3 living and 2 killed Reconstructed Human epidermis (0.5 cm2, SkinEthic RHE model) during 42 minutes, followed by

a rince with 25 mL of PBS and a 42 hours post-incubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The experimental protocol was established in accordance withO.E.C.D. test Guideline No. 439 dated 28 July 2015 and the method B.46of the Council regulation No.761/2009 dated July 23th 2009 (E.U. Journal L220) - ATP Council regulation No.440/2008 of may 30th 2008 (E.U. Journal L142).

The mean corrected percents viability of the treated tissues was 82.1%, versus 2.1%, respectively, with the positive control item (5% Sodium Dodecyl Sulfate).

In accordance with the Regulation (EC) No. 1272/2008, the test item Copper Diammine Bitetrazole (CuDABT) has to be considered as Non-irritant to skin in accordance with UN GHS No Category. No hazard statement or signal word is required.

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:

July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Provider : AUTOLIV ASP, 16700 W. Hwy 83, Promontory, UTAH 84307, USA
- batch No. P3656461
- Expiration date of the lot/batch: august 25th 2020
- Purity: 97.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in a restricted area dedicated to explosive products, at outside temperature and humidity
- Solubility and stability of the test substance in the solvent/vehicle: not soluble in water or other tested vehicle, homogeneous suspensions

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no treatment

FORM AS APPLIED IN THE TEST (if different from that of starting material)
homogeneous suspensions in vehicle

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Source: bovine eye were obtained from freshly slaughtered cattle at the abattoir EVA, Saint-Pierre-sur-Dives, FRANCE
- Age : bovine cattle were up to 12 months old

Vehicle:

other: parafin oil

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750µL of test item 20% (w/v) in vehicle (parafin oil)
homogeneous blue suspension

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

0

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): On completion of the treatment period, the test item formulation was removed from the front opening of the anterior chamber (open-chamber method) and the epithelium was rinsed as follows:
- any test item formulation adhering to the walls of the anterior chamber was removed with a cotton bud and using a pipette of heated cMEM (32°C),
- the corneas were rinsed three times with pre-warmed cMEM containing phenol red (i.e. until the test item formulation had been completely removed from the chamber or until the phenol red was not discoloured). Then, the corneas were finally rinsed with pre-warmed cMEM without phenol red.
The rinsing efficiency was visually confirmed by observing the transparency and the colour changing of the rinsing medium (containing phenol red). Some difficulties were encountered during the rinsing. Indeed, residual amounts of test item formulation were noted on the walls of the anterior chamber but were finally removed.

SCORING SYSTEM:
The following formula was used to determine the In Vitro Irritancy Score (IVIS):
IVIS = (OPT2-OPT0) + (15 x cOD490 nm)
The IVIS was calculated for each test item formulation and positive control cornea. The mean IVIS for each series of three corneas was calculated from the individual scores.


TOOL USED TO ASSESS SCORE:
OPACITY MEASUREMENTS: An opacitometer was used to measure light transmission (i.e. the level of opacity) through the center of each mounted cornea. A numerical opacity measurement (arbitrary unit) was displayed and recorded. Just before the first opacity measurement (i.e. OPT0), the opacitometer was calibrated using specific calibrators. Values obtained for each calibrator were as follows:
- calibrator No. 1: set to 75,
- calibrator No. 2: from 145 to 155,
- calibrator No. 3: from 218 to 232.
Just before the second opacity measurement (i.e. OPT2), the opacitometer was calibrated using the calibrator No. 1 set to 75. Just after each opacity measurement (OPT0 and OPT2), the calibration of the opacitometer was checked by using the calibrator No. 1. The obtained value was between 73 and 77.
Corneal opacity was determined by reading each holder in the right hand chamber of the calibrated opacitometer, versus an empty holder (without cMEM, cornea and glasses), placed in the left hand chamber.
For opacity measurement, care was taken to make sure that no air bubbles were present within the holders containing corneas (by ensuring that each compartment was filled to overflowing with heated cMEM) and each holder was wiped dry.

PERMEABILITY DETERMINATION
After the second opacity measurement, the medium of the anterior chamber was removed and the anterior chamber received 1 mL of a 5 mg/mL fluorescein solution in cMEM.
The Optical Density at a wavelength of 490 nm (OD490 nm) of this dilution was measured. As the value obtained was between 0.850 and 0.940 the fluorescein solution was validated.
For each series of three corneas, a chronometer started from the fluorescein solution application time of the first cornea of the series. The holders were incubated vertically (cornea positioned horizontally with the fluorescein-treated side uppermost) in a water bath at +32°C (± 1°C) for 90 minutes (± 5 minutes).
At the end of incubation, the maximum volume of cMEM recoverable from the posterior chamber of each holder was transferred into an identified tube. The medium was homogenized prior to determination of OD490 nm, using single-use cuvettes (1 cm path length) and a spectrophotometer (cMEM used as the blank).

MACROSCOPIC EXAMINATION
After permeability determination, the corneas were removed from the holders and observed for opaque spots, other irregularities and any separation of the epithelium (see Table 1). Then, the corneas were discarded.

Irritation parameter:

in vitro irritation score

Run / experiment:

positive control

Value:

173

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: in the range 2 SD of historical data

Irritation parameter:

in vitro irritation score

Run / experiment:

test item 20%

Value:

1

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: mean opacity <4.4 and mean fluorescein OD490nm <0.0296
- Acceptance criteria met for positive control: the mean IVIS should fall within 2 SD of the historical mean (105.2 to 186)

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test item, CuDABT, was identified as a test item not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Executive summary:

The objective of this study was to evaluate the potential irritant and corrosive properties of the test item, CuDABT, to the eye. The Bovine Corneal Opacity and Permeability (BCOP) test method can identify chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage. The design of this study was based on the guideline OECD Guideline 437 and the study was performedin compliance with CiToxLAB France standard operating procedures and with the OECD Principles of Good Laboratory Practice.

Method

Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes at +32°C.

A single experiment was performed using three corneas for each treated series (test item formulation, positive and vehicle controls).

Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer.

The test item was applied at the concentration of 20% (w/v) in the vehicle (paraffin oil), in a single experiment using a treatment time of 4 hours and using the open-chamber method. Vehicle and positive controls were applied using the same treatment time and using the closed-chamber method. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed. A second opacity measurement was then performed. After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes at +32°C. At the end of the incubation period, the optical density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities.

Results

Macroscopic examinations

No notable opaque spots or irregularities were observed on all test item formulation-treated corneas.

In Vitro Irritancy Score

All acceptance criteria were fulfilled. The study was therefore considered as valid.

The mean In Vitro Irritancy Score (IVIS) of the test item formulation-treated corneas was: 1.

As the mean IVIS was < 3, the test item was considered asa test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Conclusion

Under the experimental conditions of this study, the test item, CuDABT, was identified as a test item not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to in vitro skin irritation (reconstructed human epidermis test method, OECD 439) and in vitro skin corrosion (reconstructed human epidermis test method, OECD 431) assays results, CuDABT is not classified as irritant or corrosive for the skin :

- OECD 439: mean corrected percent viability of the treated tissues = 82.1% (>50%)

- OECD 431: 3 minutes and 1 hour after the test item application, the mean corrected percent viability of epidermis skins treated with the test item were 95.11% and 94.25% (> 50%)

According to in vitro eye irritation assay (Bovine corneal opacity and permeability test method, OECD 437), CuDABT is not classified as irritant or corrosive for the eyes:

- OECD 437: mean In Vitro Irritancy Score (IVIS) of the test item formulation-treated corneas = 1 (IVIS < 3)