Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Skin irritation (OECD 439, GLP): not irritant

(RA from CAS 6009-70-7)

Eye irritation (OECD 492, GLP): irritant

(RA from CAS 6009-70-7)

Eye irritation (OECD 437, GLP): not corrosive

(RA from CAS 6009-70-7)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 - 18 Dec 2015
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:
adopted 28 July 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
other: ECVAM 2009, Performance Standards for In-Vitro Skin Irritation Test Methods based on Reconstructed Human Epidermis (RHE)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
other: SkinEthic Skin Irritation Test-42bis Standard Operating Procedure (SOP) (2009): Using the Reconstructed Human Epidermis (RHE) model, INVITTOX Version 2.1
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Remarks:
Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: SkinEthic™ RHE-model RHE/S/17
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model (Episkin/Skin Ethic Laboratories, Lyon, France)
- Tissue batch number: 15-RHE-151
- Expiration Date: Dec 21, 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 42 ± 1 min at room temperature; thereafter at 37 °C for 42 ± 1 h

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were gently rinsed with DPBS in order to remove any residual test material. Excess DPBS was removed by gently shaking the tissue inserts and blotting the bottom of the tissues inserts with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h ± 5 min
- Spectrophotometer: microplate reader (ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the RHE model was assessed by undertaking a MTT cell viability test.
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 μL of 1% Triton X-100. The ET-50 value was determined to be 5.4 h.
- Other: Absence of significant abnormalities after histological observations (HES stained vertical paraffin)

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability is less than 50%
- The test substance is considered to be non-irritant to skin the viability is greater than or equal to 50%

ACCEPTABILITY CRITERA
The negative control OD values for the RHE-model have to be in the range of ≥ 0.8 and ≤ 3.0.
The negative control data meet the acceptance criteria if the mean OD value is higher or equal than a historically established boundary at 570 nm. The boundary is two standard deviations below the current historical mean (1.423). The standard deviation value is considered valid if ≤ 18% of the group mean value.
The positive control data meet the acceptance criteria if the mean viability value, expressed as % of the negative control, is lower than or equal to a historically established boundary. The boundary is three standard deviations above the current historical mean (3.42%). The standard deviation value is considered valid if ≤ 18% of the group mean value.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 16 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL per tissue

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL per tissue
- Concentration (if solution): 5% in deionised water
Duration of treatment / exposure:
42 ± 1 min
Duration of post-treatment incubation (if applicable):
42 ± 1 h
Number of replicates:
triplicates for each treatment and control group.
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 3 tissues
Run / experiment:
42 min exposure
Value:
99.65
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: MTT was not reduced by the test substance.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD values were 2.111, 2.004 and 2.105 and, thus, in the range of ≥ 0.8 and ≤ 3.0. The mean OD was 2.074 and the Standard Deviation was 2.90% and, thus, higher than the historically established boundary of 1.423.
- Acceptance criteria met for positive control: The mean viability value was 1.10% and the Standard Deviation was 2.22% and, thus, lower than the historically established boundary of 3.42%.
- Acceptance criteria met for variability between replicate measurements: The standard deviation of the three tissues treated with the test substance was 0.93% and, thus, ≤ 18%.
Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008
Conclusions:
Under the conditions of the conducted test, the test substance did not show irritating properties towards reconstructed human epidermis tissue. The RhE-based test methods are able to identify Cat. 2 and No Cat. chemicals and can thus serve as stand-alone skin irritation methods for non-corrosives.

CLP: not classified
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please refer to analogue justification provided in IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 3 tissues
Run / experiment:
42 min exposure
Value:
99.65
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Source: 7.3.1-1
Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008
Conclusions:
Under the conditions of the conducted test, the source substance (CAS 6009-70-7) did not show irritating properties towards reconstructed human epidermis tissue. The RhE-based test methods are able to identify Cat. 2 and No Cat. chemicals and can thus serve as stand-alone skin irritation methods for non-corrosives. Applying the RA-A approach, similar results are expected for the target substance.

CLP: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
28 Nov - 16 Dec 2016
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:
adopted in 28 July 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz
Species:
human
Strain:
other: EpiOcular™
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 50 mg
Duration of treatment / exposure:
6 h
Duration of post- treatment incubation (in vitro):
18 h
Number of animals or in vitro replicates:
in duplicates for each treatment and control group
Details on study design:
- RhCE tissue construct used, including batch number: EpiOcular™ tissue (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia), batch number: 23757
- Viability: The quality of the final tissue was assessed by undertaking an MTT cell viability test.
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 μL of 0.3% Triton X-100. The ET-50 value was determined to be 20.43 min.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: The ability of the test substance to directly reduce MTT and to form a blue/purple reaction product was assessed in a preexperiment. Since the MTT solution colour did not turn blue/purple, the test substance is not presumed to have reduced the MTT. An additional test with freeze-killed tissues did not have to be performed.
- Number of tissue replicates used per test chemical and controls: 2
- Wavelength: 570 nm
- Evaluation criteria: The test substance is considered to be not irritating to eye if the test substance-treated tissue viability is >60%.
- Acceptance criteria: The results are acceptable if the negative control OD is >0.8 and <2.5, if the mean relative viability of the positive control is below 50% of the negative control viability and if the difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items).
- Reference to historical data of the RhCE tissue construct: Historical control data was used to assess the validity of the test.
Irritation parameter:
other: % tissue viability mean value of 2 tissues
Run / experiment:
6 h exposure
Value:
3.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER OBSERVATIONS:
The optical pre-experiment (colour interference pre-experiment) to investigate the colour change potential of the test substance in water or isopropanol did not lead to a change in colour. Optical evaluation of the MTT-reducing capacity of the test substance with MTT-reagent did not show blue colour.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD values between 1.361 and 1.417 was in the range of > 0.8 and < 2.5.
- Acceptance criteria met for positive control: Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 18.2%, thus the validity of the test system is ensured.
The difference of viability between the two relating tissues of a single substance is < 20% (values between 1.3% to 4.6%) in the same run (for positive and negative control tissues and tissues of single test substance).

Table 1: Results after treatmen for 6 h with the test substance and the controls

Dose Group Absorbance Well 1 (Tissue 1/2) Absorbance Well 2 (Tissue 1/2) Mean Absorbance (Tissue 1/2) Mean Absorbance* Tissue 1 and 2 minus Mean Blank Mean Absorbance of 2 Tissues* Rel. Absorbance [%] Tissue 1 and 2** Difference of the Rel. Absorbances [%] Tissue 1 and 2 Viability
[% of Negative Control]**
Blank 0.038 0.038 0.038 0.000        
Negative Control 1.384 1.361 1.373 1.335 1.355 98.5 3.0 100.0
1.417 1.410 1.414 1.376 101.5
Positive Control 0.249 0.258 0.253 0.215 0.247 15.9 4.6 18.2
0.318 0.314 0.316 0.278 20.5
Test substance 0.072 0.072 0.072 0.034 0.042 2.5 1.3 3.1
0.089 0.089 0.089 0.051 3.8

Table 2 Historical Control Data

Positive Control Negative Control [OD570]
Mean Viability 32.47% Mean Absorption 1.49
Rel. Standard Deviation 10.29% Rel. Standard Deviation 0.24
Range of Viabilities 15.90% - 42.30% Range of Absorbance 1.24 - 2.05
Mean Absorption 0.48  
Rel. Standard Deviation 0.14
Range of Absorbance 0.22- 0.64
Interpretation of results:
other: the results of this study as a stand-alone study are not suitable for classification according to CLP/EU GHS criteria; the results may be used for classification purposes in a weight of evidence approach
Conclusions:
Under the conditions of the conducted EpiOcular test, the test substance showed irritating properties towards human-derived epidermal keratinocytes in the EpiOcular™ model.
Based on the weight of evidence approach in consideration of the results of the BCOP Assay (please refer to reference 7.3.2-2) and the Epiocular test (reference 7.3.2-1), the test substance meet the classification criteria as eye irritant according to Regulation(EC) No. 1272/2008.

CLP: Eye Irrit. Cat. 2, H319
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
15 Jan 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 Ocular Corrosives and Severe Irritants)
Version / remarks:
adopted 23 July 2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Odenwaldschlachthof Brensbach, Brensbach, Germany
- Characteristics of donor animals: 14 – 36 months old
- Storage, temperature and transport conditions of ocular tissue: The freshly isolated eyes were transported in transport medium on ice.
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes and directly used in the BCOP test.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20% (w/v)

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 0.9% sodium chloride solution
Duration of treatment / exposure:
240 min
Duration of post- treatment incubation (in vitro):
90 min
Number of animals or in vitro replicates:
triplicates for each treatment and control group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS:
The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 to 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea. Each cornea was mounted in a specially designed cornea holder.

QUALITY CHECK OF THE ISOLATED CORNEAS:
At the end of the equilibration period, the basal opacity was determined (t0). Each cornea with a value of a basal opacity >7 was discarded.

TREATMENT METHOD:
The cornea holder consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O- ring without stretching the cornea. The anterior part of the holder was positioned on the top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. The anterior compartment received the test substance or the controls on the surface of the corneae. The corneae were incubated in a horizontal position at 32 ± 1 °C.

REMOVAL OF TEST SUBSTANCE:
The test substance was rinsed off from the application side with wash medium.
- POST-EXPOSURE INCUBATION: 90 min in a vertical position

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany).
- Corneal permeability: The passage of sodium fluorescein dye was measured with the aid of a microplate reader (ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany) at 490 nm.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as severe irritant/corrosive and labelled Category 1.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; ≤ 55: no prediction can be made.
Irritation parameter:
in vitro irritation score
Run / experiment:
240 min exposure
Value:
9
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
With the negative control (0.9% (w/v) NaCl solution) neither an increase of opacity nor permeability of the corneae was observed, the calculated IVIS was 1.1 and thus, within three standard deviations of the current historical mean of the negative control (IVIS: -1.3 – 3.5). The positive control (imidazole) showed clearly increased opacity and distinctive permeability of the corneae fulfilling the criteria as severe irritating/corrosive. The calculated IVIS was 100.2 and thus, also within the standard deviations of the current historical mean of the positive control (IVIS 75.9 – 136.9).
The IVIS obtained after treatment with the test substance was 9.0 and thus no prediction can be made regarding the eye hazard potential of the test substance.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control resulted in an IVIS score of 1.1, which was within three standard deviations of the current historical mean of the negative control.
- Acceptance criteria met for positive control: The positive control resulted in an IVIS score of 100.2, which was within two standard deviations of the current historical mean of the positive control.

Table 1: Summary of results

  Opacity Permeability IVIS
per cornea per group Standard Deviation
Negative Control 2.895 0.004 2.955 1.1 1.7
0.000 0.001 0.10
0.197 0.000 0.192
Positive Control 63.314 2.305 97.883 100.2 2.6
66.788 2.195 99.711
63.494 2.640 103.098
Test substance  8.819 0.036 9.352 9.0 0.3
8.678 0.028 9.097
8.391 0.019 8.675

Table 2: Historical control data

  Opacity Permeability IVIS
per cornea per group Standard Deviation
Negative Control 1.133 -0.001 1.123 1.2 1.1
0.188 -0.001 0.178
1.877 0.029 2.307
Positive Control 47.347 2.292 81.725 91.2 11.1
62.827 1.710 88.480
59.002 2.955 103.330
Interpretation of results:
other: the results of this study as a stand-alone study are not suitable for classification according to CLP/EU GHS criteria; the results may be used for classification purposes in a weight of evidence approach
Conclusions:
Under the conditions of the conducted test, a prediction regarding the eye hazard potential according to OECD 437 after exposure to the test substance is not possible.
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
Summary of available data used for the endpoint assessment of the target substance.
Adequacy of study:
weight of evidence
Justification for type of information:
Please refer to analogue justification provided in IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Irritation parameter:
other: % tissue viability mean value of 2 tissues
Run / experiment:
6 h exposure
Value:
3.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Source: 7.3.2-1
Irritation parameter:
in vitro irritation score
Run / experiment:
240 min exposure
Value:
9
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: Source: 7.3.2-2
Interpretation of results:
other: the results of either of these studies as stand-alone studies are not suitable for classification according to CLP/EU GHS criteria; the results may be used for classification purposes in a weight of evidence approach
Conclusions:
Under the conditions of the conducted EpiOcular test, the source substance (CAS 6009-70-7) showed irritating properties towards human-derived epidermal keratinocytes in the EpiOcular™ model.
Based on the weight of evidence approach in consideration of the results of the BCOP Assay (please refer to reference 7.3.2-2) and the Epiocular test (reference 7.3.2-1), the source substance (CAS 6009-70-7) meet the classification criteria as eye irritant according to Regulation(EC) No. 1272/2008. Applying the RA-approach, similar results are expected for the target substance.

CLP: Eye Irrit. Cat. 2, H319
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for read-across

There are no reliable data available regarding irritation and corrosion for either dipotassium oxalate monohydrate (CAS 6487-48-5) or dipotassium oxalate anhydate (CAS 583-52-8). Read-across from an appropriate substance (diammonium oxalate monohydrate (CAS 6009-70-7) is conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5. in order to fulfil the standard data requirements defined in Regulation (EC) No 1907/2006, Annex VII, 8.1 and 8.2. Common functional groups, structural similarities and comparable toxicological properties (according to the joint consideration in Annex VI to CLP) of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

Skin irritation

CAS 6009-70-7

The skin irritancy potential of the source substance diammonium oxalate monohydrate was determined in an in vitro skin irritation study using a human skin model according to OECD TG 439 and GLP (reference 7.3.1-1). In this study the test substance did not show any irritating properties (tissue viability of 99.65%) towards human-derived epidermal keratinocytes.

 

Eye irritation

CAS 6009-70-7

The eye irritation potential of the source substance diammonium oxalate monohydrate was determined by an in vitro eye irritation test using a human cornea model according to OECD Guideline 492 and in compliance with GLP (reference 7.3.2-1). In this study the test substance showed irritating properties towards human-derived epidermal keratinocytes.

To exclude corrosive properties towards the eyes, a bovine corneal opacity and permeability (BCOP) test according to OECD Guideline 437 was conducted in compliance with GLP with the source substance diammonium oxalate monohydrate (reference 7.3.2-2). Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 9. According to OECD Guideline 437, the test substance does not have to be classified as corrosive.

In conclusion, based on the available results, the test substance is considered to show irritating properties towards the eyes.

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to dipotassium oxalate, data will be generated from information on reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

The available data on skin irritation from the source substance diammonium oxalate do not meet the criteria for classification according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification. The available data on eye irritation from the source substance diammonium oxalate meet the criteria for classification as Eye Irrit. Cat. 2 (H319) according to Regulation (EC) 1272/2008.

Therefore, applying the RA-A approach, the target substance dipotassium oxalate is also considered not to meet the classification criteria for skin irritation and to meet the criteria for classification as Eye Irrit. Cat. 2 (H319) according to Regulation (EC) 1272/2008.