Reaction mass of diammonium (2R,3R)-2,3-dihydroxybutanedioate and diammonium bis[(2R,3R)-2,3-dioxidobutanedioato]diantimonate(2-)

Endpoint summary

Description of key information

A GLP-compliant in vitro skin irritation and corrosion turnkey testing strategy was conducted in the EpiDerm Test according to OECD Guideline 431 and OECD Guideline 439 to assess the skin irritating / corrosion potential of the test substance. Based on the results observed in the studies the test substance shows a skin irritation potential in the EpiDerm in vitro skin irritation and corrosion test strategy.

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:

Jan - Feb 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(from the competent authority) Landesamt für Umwelt Rheinland-Pfalz

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 170492F01
- Expiration date of the Batch: 22 Jun 2019
- Purity: 100.0 area-% (HPLC fingerprint)
- Content:
21.4 g/100 g Test Item
23.5 g/100 g (NH4)2C4H4O6
30 g/100 g Tartrate
6.7 g/100 g Ammonium
9.1 g/100 g Antimony
53.5 g/100 g Water
- pH value: ca. 9 (undiluted test substance)
- Physical state / color: Liquid / colorless to yellowish, clear

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

FORM AS APPLIED IN THE TEST: undiluted liquid test substance

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: EPI-200
- Tissue Lot-number: 25882

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: two

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Incubation time: 3 h
- Spectrophotometer: Sunrise Absorbance Reader
- Wavelength: 570 nm
- Filter: measurement without reference filter

NUMBER OF REPLICATE TISSUES: 2

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%.

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 and 1 h

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure period

Value:

85.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 h exposure period

Value:

79.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Table 1: Exposure period 3 min: Individual and mean OD570 values, individual and mean viability values, standard deviations and coefficient of variation

Test substance indetification		Tissue 1	Tissue 2	Mean	SD	CV [%]
NC	Mean OD570	1.789	1.779	1.784
	Viability [% of NC]	100.3	99.7	100.0	0.4	0.4
Test substance	Mean OD570	1.516	1.534	1.525
	Viability [% of NC]	85.0	86.0	85.5	0.7	0.8
PC	Mean OD570	0.179	0.185	0.182
	Viability [% of NC]	10.0	10.4	10.2	0.2	2.3

NC: negative control

PC: positive control

SD: standard deviation

CV: coefficient of variation

Table 2: Exposure period 1 h: Individual and mean OD570 values, individual and mean viability values, standard deviations and coefficient of variation

Test substance indetification		Tissue 1	Tissue 2	Mean	SD	CV [%]
NC	Mean OD570	1.479	1.668	1.574
	Viability [% of NC]	94.0	106.0	100.0	8.5	8.5
Test substance	Mean OD570	1.200	1.294	1.247
	Viability [% of NC]	76.2	82.3	79.3	4.2	5.4
PC	Mean OD570	0.065	0.089	0.077
	Viability [% of NC]	4.1	5.7	4.9	1.1	22.5

NC: negative control

PC: positive control

SD: standard deviation

CV: coefficient of variation

Interpretation of results:

study cannot be used for classification

Conclusions:

No prediction can be made for skin corrosion according to GHS criteria based on the results of this in vitro study alone.

Executive summary:

The potential of the test substance to cause dermal corrosion was assessed by a single topical application of 50 µL undiluted test substance to a reconstructed three-dimensional human epidermis modell (EpiDerm).

Two EpiDerm tissues were incubated with the test substance for 3 minutes and 1 hour, each. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure / post-incubation by using a colorimetric test. The reduction of mitochondrial tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The test substance is not able to reduce MTT directly.

The relative mean viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was 85.5 %, and it was 79.3 % after an exposure period of 1 hour.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Jan - Feb 2018

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

GLP compliance:

yes (incl. QA statement)

Remarks:

(from the competent authority) Landesamt für Umwelt Rheinland-Pfalz

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 170492F01
- Expiration date of the Batch: 22 Jun 2019
- Purity: 100.0 area-% (HPLC fingerprint)
- Content:
21.4 g/100 g Test Item
23.5 g/100 g (NH4)2C4H4O6
30 g/100 g Tartrate
6.7 g/100 g Ammonium
9.1 g/100 g Antimony
53.5 g/100 g Water
- pH value: ca. 9 (undiluted test substance)
- Physical state / color: Liquid / colorless to yellowish, clear

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

FORM AS APPLIED IN THE TEST: undiluted liquid test substance

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: EPI-200
- Tissue Lot number: 25876

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature for 25 min and 37 °C for 35 min
- Temperature of post-treatment incubation (if applicable): 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 1

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Incubation time: 3 h
- Spectrophotometer: Sunrise Absorbance Reader
- Wavelength: 570 nm
- Filter: measurement without reference filter

NUMBER OF REPLICATE TISSUES: three

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the mean percent tissue viability after exposure and post-treatment incubation is less than or equal to 50 %.

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

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL

Duration of treatment / exposure:

1 h

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour exposure

Value:

2.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Table 1: Individual and mean OD570 values, individual and mean viability values, standard deviations and coefficient of variation

Test substance identification		Tissue 1	Tissue 2	Tissue 3	Mean	SD	CV [%]
NC	Mean OD570	1.791	1.704	1.982	1.826
	Viability [% of NC]	98.1	93.3	108.6	100.0	7.8	7.8
Test substance	Mean OD570	0.046	0.057	0.058	0.054
	Viability [% of NC]	2.5	3.1	3.2	2.9	0.4	12.9
PC	Mean OD570	0.040	0.039	0.040	0.039
	Viability [% of NC]	2.2	2.1	2.2	2.2	0.0	1.5

NC: negative control

PC: positive control

SD: standard deviation

CV: coefficient of variation

Interpretation of results:

study cannot be used for classification

Conclusions:

No prediction can be made for skin irritation according to GHS criteria based on the results of this in vitro study alone.

Executive summary:

The potential of the test substance to cause dermal irritation was assessed by a single topical application of 30 µL undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm).

The test was performed with three EpiDerm tissues, which were incubated with the test substance for 1 hour followed by a 42 -hour post-incubation period.

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

The test substance is not able to reduce MTT directly.

The relative mean viability of the tissues treated with the test substance determined after an exposure period of 1 hour with an about 42 -hour post-incubation was 2.9 %.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation Turnkey Testing Strategy

The objective was to assess the skin irritation and corrosion potential of the test substance. Using the methods currently available, a single in vitro assay is not sufficient to cover the full range of skin irritating / corrosion potential. Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT).

The potential of the test substance to cause dermal corrosion / irritation was assessed by a single topical application of 50 µL (corrosion test) or 30 µL (irritation test) undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm).

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

Tissues destruction was determined by measuring the metabolic activity of the tissue after exposure / post-incubation by 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 epidermal tissues treated with the test substance is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The test substance is not able to reduce MTT directly.

Results of the Corrosion Test (SCT): The relative mean viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was 85.5 %, and it was 79.3 % after an exposure period of 1 hour.

Results of the Irritation Test (SIT): The relative mean viability of the tissues treated with the test substance determined after an exposure period of 1 hour with an about 42 -hour post-incubation was 2.9 %.

Based on the results observed and by applying the evaluation criteria, it was concluded that the test substance shows a skin irritation potential in the EpiDerm in vitro skin irritation and corrosion test strategy under the test conditions chosen.

Eye Irritation Turnkey Testing Strategy

The objective was to assess the eye irritating potential of the test substance. By using the methods currently available a single in vitro assay is not sufficient to cover the full range of eye irritating potential. Therefore, two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular Eye Irritation Test.

BCOP:

The potential of the test substance to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 µL 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 -hour post-incubation period. In addition to the test substance, a negative control (NC, deionized water) and a positive control (PC, 100 % ethanol) were applied to three corneas each. Corneal opacity was quantitatively measured as the amount of light transmitted through the cornea. Permeability was quantitatively measured 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 of the test substance. The result of the mean IVIS for the test substance was determined to be 4.9.

EpiOcular:

The potential of the test substance to cause ocular irritation was assessed by a single topical application of 50 µL undiluted test substance to a reconstructed three-dimensional, human cornea model (EpiOcular). Six test runs were performed. Two of them did not reveal valid results and are not reported. Two EpiOcular tissues per test run were incubated with the test substance for 30 minutes followed by a 2 -hour post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure / post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation wth a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance 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 eye irritation assay: The test substance is not able to directly reduce MTT.

1st test run: The relative mean viability of the tissues treated with the test substance was 60.8 % (values for single tissues: 64.9 % and 56.7 %). Due to the borderline result another test run of the EpiOcular eye irritation test was performed.

2nd test run: The relative mean viability of the tissues treated with the test substance of the 2nd test run was 53.5 % (values for single tissues: 50.3 % and 56.6 %). Due to the borderline results obtained after both test runs, a 3rd test run was performed to increase the database for final evaluation.

3rd test run: The relative viabilities of the single tissues treated with the test substance of the 3rd test run was 21.2 % and 70.9 %. Due to high inter-tissue variability (49.8 %) of the test substance treated tissues, the acceptance criteria was not met. Thus, the study was repeated.

No valid results were obtained within the 4th and 5th test run, because the acceptance criteria of the negative control were not met.

6th test run: The relative mean viability of the tissues treated with the test substance of the 6th test run was 72.5 % (values for single tissues: 84.2 % and 60.8 %). The value for inter-tissue variability of the test substance (23.4 %) was slightly out of the acceptance range. However, no further test run was performed.

The result of the BCOP Test alone does not allow for evaluation of eye irritation. On basis of the result of this study, a potential of the test substance to bear a risk of eye irritation cannot be excluded. The assessment of the EpiOcular eye irritation assay showed that no unambiguous result could be derived within four test runs. In summary, it must be concluded that the results of the test substance should be considered as "inconclusive" in the in vitro eye irritation test strategy under the test conditions chosen. For final assignment of a risk phrase at present, results from another study would be needed.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. A GLP-compliant turnkey testing strategy including the EpiDerm skin corrosion test (SCT, OECD Guideline 431) and the EpiDerm skin irritation test (SIT, OECD Guideline 439) is available for skin irritation. In the skin corrosion test, the score for the treated tissues indicates that the test substance is a non-corrosive. The result of the skin irritation test demonstrates that the test substance has to be classified for Cat. 2 or Cat. 1. Therefore it can be concluded that the test item is considered to be classified for skin irritation Cat. 2 under Regulation (EC) No. 1272/2008, as amended for the tenth time in Regulation (EC) No. 2017/776.