Tetraamminezinc(2+) carbonate

Endpoint summary

Description of key information

Skin irritation/corrosion: Based on an in vitro test strategy the test item shows a skin irritation potential.

Eye irritation: Based on an in vitro test strategy the test item shows an eye irritation potential.

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:

weight of evidence

Study period:

9 December 2016 - 17 November 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)

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: B.40 BIS.: Methods for the determination of toxicity and other health effects: In Vitro Skin Corrosion: Human Skin Model Test; Official Journal of the European Union, No. L 142

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: IATA for skin corrosion and irritation, Series on Testing and Assessment No. 203

Version / remarks:

11 July 2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Kampagne 02/2014
- Expiration date of the lot/batch: February 18, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ 200 kit ( EPI-200 tissues with surface of 0.6 cm²), MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Tissue lot number: 23385

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 min at room temperature or 1 hour in the incubator at 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing: with sterile PBS

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg / mL
- Incubation time: 3 hours
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS
- Viability: 1.404 ± 0.016
- Barrier function: ET-50 = 6.44 hours
(Lower acceptance limit: ET50 = 4.0 hours and Upper acceptance limit: ET50 = 8.7 hours)
- Morphology: functional stratum corneum, a viable basal cell layer, intermediate spinous and granular layers
- Contamination: no, sterile

NUMBER OF REPLICATE TISSUES: 2 per exposure time

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues: EPI-200 tissue that is killed by freezing at –20°C
- N. of replicates : 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2

PREDICTION MODEL / DECISION CRITERIA
Step 1: Identification of corrosives:
mean tissue viability
(% of negative control) Prediction
< 45 after 3 min exposure corrosive
>45 after 3 min exposure and < 10% after 1h exposure corrosive
45-55 after 3 min exposure borderline (inconclusive)1
> 55% after 3 min exposure and < 10-20% after 1h exposure borderline (inconclusive) 1
> 55% after 3 min exposure and > 20% after 1 hour exposure non-corrosive
1 The borderlines-evaluation (50 ± 5%, 25 ± 5% and 15 ± 5%) was determined statistically using historic BASF data and hence considers the variance of the test method. This evaluation is an amendment to the evaluation provided in OECD Guideline 431.

Step 2: Optional UN GHS subcategorization of corrosives identified in step 1:
% of negative control) Prediction
< 20 after 3 min exposure UN GSH Cat 1A
20-30% after 3min exposure borderline (inconclusive) for UN GHS subcategorization
> 30% after 3 min exposure UN GHS Cat 1B or 1C
According to the current OECD Guideline 431 differentiation of UN GHS categories 1A versus 1B/C is possible. However, the subcategorization into UN GHS Cat 1A is over-predictive (29% of 1B/C substances are over-predicted as 1A) as stated in the Guideline and differentiation between GHS sub-categories 1B or 1C is not possible. If the test substance is identified to be corrosive by SCT and a transport classification is needed an additional test appropriate for UN GHS subcategorization should be performed to confirm classification as UN GHS Cat 1A or to differentiate between UN GHS Cat 1B and 1C.

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

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

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

Duration of treatment / exposure:

3 min or 1 hour

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1st run (3 min)

Value:

91.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

12.4%

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1st run (1 h)

Value:

20.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

5.6%

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2nd run (1 h)

Value:

22.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

6.2%

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: yes
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
Tissue viability is acceptable if the mean OD570 of the NC is ≥ 0.8. The mean OD570 of the NC should not exceed 2.8.
- Acceptance criteria met for positive control: yes
A tissue viability of ≤ 30% is acceptable for the 3-minute exposure. Mean viability of the tissues exposed for 1 hour should be <15%.
- Acceptance criteria met for variability between replicate measurements: yes
In the range of 20% and 100% viability, variability between the tissues is considered to be acceptable if the coefficient of variation (CV) of %-viability is ≤ 30%.

Historic control data:
Historic Range of NC (Period Jan 2015- Jan 2017)
3 min exposure: Mean OD570 1.919 ± 0.177
60 min exposure: Mean OD570 1.936 ± 0.193

Historic Range of PC (Period Jan 2015- Jan 2017)
3 min exposure: Mean OD570 0.282 ± 0.073
60 min exposure: Mean OD570 0.121 ± 0.033

Viability % (Period Jan 2015- Jan 2017)
3 min exposure: Mean % 14.7 ± 3.8
60 min exposure: Mean % 6.2 ± 1.4

Interpretation of results:

other: not skin corrosive (Cat. 1)

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

9 December 2016 - 17 November 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)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

6 July 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: IATA for skin corrosion and irritation, Series on Testing and Assessment No. 203

Version / remarks:

11 July 2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Kampagne 02/2014
- Expiration date of the lot/batch: February 18, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ 200 kit ( EPI-200 tissues with surface of 0.6 cm²), MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Tissue lot number: 23385

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 25 min at room temperature and 35 min in the incubator at 37°C
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing: with sterile PBS
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg / mL
- Incubation time: 3 hours
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS
- Viability: 1.404 ± 0.016
- Barrier function: ET-50 = 6.44 hours
- Morphology: functional stratum corneum, a viable basal cell layer, intermediate spinous and granular layers
- Contamination: no, sterile

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues: EPI-200 tissue that is killed by freezing at –20°C
- N. of replicates : 3

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

PREDICTION MODEL / DECISION CRITERIA
mean tissue viability
(% of negative control) Prediction
< 45 irritant
45 - 55 borderline
> 55 non-irritant
The „borderline“-evaluation (50 ± 5%) was determined statistically using historic BASF data and hence considers the variance of the test method.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 30 µL

NEGATIVE CONTROL
- Amount(s) applied: 30 µL

POSITIVE CONTROL
- Amount(s) applied: 30 µL
- Concentration: 5% (w/v) sodium dodecyl sulfate (SDS) in water

Duration of treatment / exposure:

1 hour

Duration of post-treatment incubation (if applicable):

ca. 42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

1.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100 %

Positive controls validity:

valid

Remarks:

2.5 %

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECT
- Direct-MTT reduction: yes
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
Tissue viability is acceptable if the mean OD570 of the NC is ≥ 0.8. The mean OD570 of the NC should not exceed 2.8.
- Acceptance criteria met for positive control: yes
A viability of ≤ 20% is acceptable.
- Acceptance criteria met for variability between replicate measurements: yes
The inter-tissue variability is considered to be acceptable if the SD of %-viability is ≤ 18%.

Historic control data of PC and NC:
Historic Range of NC (Period Jan 2014 - Nov 2016)
Mean OD570: 2.272 ± 0.297

Historic Range of PC (Period Jan 2014 - Nov 2016)
Mean OD570: 0.069 ± 0.012

Viability (%) (Period Jan 2014 - Nov 2016)
Mean % : 3.1 ± 0.5

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

14 December 2016 - 17 November 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)

Version / remarks:

26 July 2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Kampagne 02/2014
- Expiration date of the lot/batch: February 18, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Schlachthof Mannheim, Schlachthofstr. 21, 68165 Mannheim, Germany
- Characteristics of donor animals: freshly slaughtered cattle, age of the animals: minimum 12 months, maximum 60 months

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagle’s MEM (without phenol red) and then equilibrated in a vertical position at about 32°C for at least 1 hour. After the equilibration period the medium in both chambers was replaced with fresh prewarmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer.

QUALITY CHECK OF THE ISOLATED CORNEAS
Any corneas that showed macroscopic tissue damage or an opacity value < 550 opacity units were discarded.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: de-ionized water

POSITIVE CONTROL USED: 100% ethanol

APPLICATION DOSE AND EXPOSURE TIME
Before application, the medium in the anterior chamber was removed using a syringe. 750 μL of the undiluted liquid test substance was applied into the anterior chamber using a pipette. The corneas were incubated in a horizontal position at about 32 °C for approximately 10 minutes.

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red)
- POST-EXPOSURE INCUBATION: 2 hours at about 32 °C

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others: before measurement, each cornea was observed visually and observations were recorded

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITARIA:
The following decision criteria apply:

IVIS Prediction
< 1.5 No classification for eye irritation (1)
1.5 – 4.5 Borderline (1,3)
> 4.5; < 45 No prediction can be made for eye irritation, further testing with another suitable method is required (1,2)
45 - 65 Borderline (1,3)
> 65 Ocular corrosive or severe irritant
(1) According to OECD Guideline 437 (adopted July 2013), this prediction is possible, however, due to limited accuracy of the BCOP test to correctly identify substances that do not require classification for eye irritation or serious eye damage, this test method should not be the first choice to initiate a bottom-up approach. Based on this statement and the experience of the test facility, test substances not leading to the prediction “ocular corrosive or severe irritant” are
generally examined in the EpiOcular test as well.
(2) The test method according to the OECD Guideline 437 revised and adopted in 2013 does not allow for the evaluation of eye irritation. I.e., the result does not exclude an irritation potential of the test substance. For final assignment of a risk phrase at present, results from another study are needed.
(3) The borderline“-evaluation (IVIS 3.0 ± 1.5 and 55.0 ± 10.0) was determined statistically using historic BASF data and takes the test facility specific variance of the test method into account. This evaluation is an amendment to the evaluation provided in OECD Guideline 437.

Irritation parameter:

cornea opacity score

Run / experiment:

1st experiment

Value:

23.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

2.4

Positive controls validity:

valid

Remarks:

24.1

Irritation parameter:

other: permeability score

Run / experiment:

1st experiment

Value:

0.067

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0

Positive controls validity:

valid

Remarks:

0.931

Irritation parameter:

in vitro irritation score

Run / experiment:

1st experiment

Value:

24.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

2.4

Positive controls validity:

valid

Remarks:

38 (PC1) and 107.4 (PC2)

Interpretation of results:

other: not eye damaging (Cat.1)

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

14 December 2016 - 17 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Kampagne 02/2014
- Expiration date of the lot/batch: February 18, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.

Details on test animals or tissues and environmental conditions:

- Description of the cell system used:
The EpiOcularTM model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinocytes used to model the human corneal epithelium. The EpiOcularTM tissues (surface 0.6 cm²) are cultured on cell culture inserts and are commercially available as kits (EpiOcular™200), containing 24 tissues on shipping agarose.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 µL

Duration of treatment / exposure:

30 min

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

2

Details on study design:

- RhCE tissue construct used, including batch number:
Tissue model: OCL-200
Tissue Lot Number: 23758
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava,Slovakia

- Doses of test chemical and control substances used:
50 µL of NC (de-ionized water) and PC (Neat methyl acetate)

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
30 min exposure at 37°C in the incubator; 2 hours post-exposure at 37°C in the incubator

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled): 2

- Description of the method used to quantify MTT formazan:
The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically (Spectrophotometer: SunriseTM Absorbance Reader)

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
Mean tissue viability Prediction
(% of negative control)
< 55 Irritant
55 - 65 Borderline
> 65 Non-irritant
The „borderline“-evaluation (60 ± 5%) was determined statistically using historic BASF data and hence considers the variance of the test method. This evaluation is an amendment to the evaluation provided in OECD Guideline 492.

- Positive and negative control means and acceptance ranges based on historical data
Historic Range of NC
OD570( Period Jan 2014 - Nov 2016, liquids)
Mean OD 1.931 ± 0.263

Historic Range of PC
OD570( Period Jan 2014 - Nov 2016, liquids)
Mean OD 0.572 ± 0.144

- Acceptable variability between tissue replicates for positive and negative controls:
NC: The absolute OD570 of the NC-tissues in the MTT-test is an indicator of tissue viability obtained in the testing
laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is > 0.8. The mean OD570 of the NC should not exceed 2.5.
PC: Methyl acetate used as PC usually leads to a tissue viability of approx. 25%. A viability of < 50% is acceptable.

- Acceptable variability between tissue replicates for the test chemical:
Two tissues were treated under the same conditions. A variability between the two tissues is considered to be acceptable if the relative difference of the viability is < 20%.

Irritation parameter:

other: % viability

Run / experiment:

1st experiment

Value:

4.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

24.8%

Other effects / acceptance of results:

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

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion

The potential of the test item to cause dermal corrosion/irritation was assessed in two in vitro assays as part of an in vitro skin irritation and corrosion test strategy. Both tests were performed according OECD test guidelines (OECD TG 431 and OECD TG 439)

The test item was applied by a single topical application of 50 μL (corrosion test) or 30 μL (irritation test) of the undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm TM).

For the corrosion test two EpiDerm (TM) tissues were incubated with the test substance for 3 minutes and 1 hour, each. The irritation test was performed with three EpiDerm (TM) tissues, which were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test-substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The test substance is able to reduce MTT directly. Therefore an additional MTT reduction control KC (freeze-killed control tissues) was introduced.

Results of the Corrosion test:

The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 91.6%, and it was 20.4% after an exposure period of 1 hour. Due to the borderline result obtained at the 1-hour exposure period (values for single tissues:19.0% and 21.7%), another test run of the 1-hour exposure was performed. At the second test run the mean viability of the test-substance treated tissues determined after an exposure period of 1 hour was 22.2% (values for single tissues: 20.8% and 23.6%). The mean viability value of both test runs is about 21.3% and thus lies above the cut off for skin corrosion.

Results of the Irritation test:

The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 1.8%.

Based on the observed results it was concluded that the test item shows a skin irritation potential in the EpiDerm (TM) in vitro skin irritation and corrosion test strategy under the test conditions chosen (BASF, 2017)

Eye irritation

The potential of the test item to cause eye irritation was assessed in two in vitro assays as part of an in vitro eye irritation test strategy. The tests were performed according OECD TG 437 (BCOP test) and OECD TG 492 (EpiOcular).

BCOP test

The potential of the test item to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 50μL of the undiluted test substance to the epithelial surface of isolated bovine corneas.Three corneas were treated with the test substance for 10 minutes followed by a 2-hours postincubation period. In addition to the test substance a negative control (NC; de-ionized water) and positive controls (PC1 / PC2; 100% ethanol / 100% dimethylformamide) were applied to three corneas each.

Corneal opacity was measured quantitatively as the amount of light transmitted through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score (IVIS) of the test substance.

An IVIS of 24.4 was reported for the test item. For the NC and PC an IVIS of 2.4 and 38.1 (PC1) or 107.4 (PC2) was detected, respectively.

EpiOcular

The potential of the test item to cause ocular irritation was assessed by a single topical application of 50 μL of the undiluted test substance to a reconstructed three-dimensional human cornea model (EpiOcular). Two EpiOcular (TM) tissues were incubated with the test substance for 30 minutes followed by a 2-hours post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test-substance treated epidermal tissues is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability.

The following results were obtained in the EpiOcular (TM) eye irritation assay:

The test substance is able to reduce MTT directly. Therefore an additional MTT reduction control KC (freeze-killed control tissues) was introduced.

The mean viability of the test-substance treated tissues was 4.2%.

 

Based on the results for BCOP and EpiOcular Test, it was concluded that the test item shows an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen (BASF, 2017).

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No 1272/2008. Based on this information the test item is considered to be classified for skin irritation Cat. 2 (H315: "Causes skin irritation") and eye irritation Cat. 2 (H319: "Causes serious eye irritation") under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.