Disodium 1,3,4-thiadiazole-2,5-dithiolate

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-01-10 - 2017-03-09 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
see target record

Cross-reference

Reason / purpose for cross-reference:

read-across source

Remarks:

link to target

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2017-01-10 - 2017-03-09 (experimental phase)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Klimisch 1 source record, but performed on read-across substance

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
General remarks ahead:
The substance is unstable when isolation from water and amine stabilizers and / or manufacture without water is attempted. The solid DMTD-Na is, based on handling observations, only stable as aqueous solution in combination with stabilizers as set out in IUCLID section 1.2. As a pure substance, it is highly unstable towards oxygen (after contact the corresponding dimer is formed), and very hygroscopic. In consequence, the substance needs to be registered including water (required for stabilization) and water is hence part of the substance ID. The substance should still be regarded as a mono-constituent substance according to the “Guidance for identification & naming of substances under REACH & CLP” (v1.3). Analytics revealed that the substance is composed of Disodium 1,3,4-thiadiazole-2,5-dithiolate (CAS 55906-42-8), water and stabilizing amines, no other DMTD-species was found. This is comparable to the structurally related read-across substance DMTD (1,3,4-Thiadiazole-2,5-dithiol, CAS 1072-71-5). Water is toxicologically irrelevant, and so, only the similarities between the two solid substances need to be regarded.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across is justified out of the following reasons:
1. The substances are structural analogues. The registered substance is produced from a neutralization reaction of 1,3,4-Thiadiazole-2,5-dithiol (CAS 1072-71-5, EC 214-014-1; source) and Sodium hydroxide (CAS 1310-73-2, EC 215-185-5) in the presence of stabilizers. So the organic ring remains intact. During analysis of DMTD-Na via HPLC, it cannot even be detected anymore but is transformed in the source substance. So the prerequisites of common functional group(s) and common precursor(s) are fulfilled.
2. The source and target substance exhibit both similar properties, or the source substance properties can be regarded as worst case, while the target substance bear toxicologically less relevant properties, see point 4. Data Matrix.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
For the properties of the target substance, please refer to section 1.2
Property Source chemical Target Chemical
CAS RN 1072-71-5 55906-42-8
Name 1,3,4-Thiadiazole-2,5-dithiol Disodium 1,3,4-thiadiazole-2,5-dithiolate
EC-No. 214-014-1 259-886-4
Other contained substances None; substance is pure Water
Stabilizing amines (see section 1.2), either not classified or not contained to a relevant amount
In both substances, only the substance was found analytically. There was no indication that possibly dimers or other oligomeric substances are present.

3. ANALOGUE APPROACH JUSTIFICATION
First, the substances are structural analogues. They have common functional group(s) and common precursor(s). Second, the impurity profile does not hinder the read-across. The source chemical is pure, and the target substance does only contain other substances which are not relevant for classification. They have the same basic structure, no dimers or oligomers are found. Third, during analysis in a slightly acidic, aqueous eluent, which is relevant for body fluids, too, the registered substance was even transformed back into the source chemical. Fourth, they have the same toxicological properties, based on the actually available information, see 4. Data matrix.

4. DATA MATRIX
Endpoint Source chemical Target Chemical
CAS RN 1072-71-5 55906-42-8
Name 1,3,4-Thiadiazole-2,5-dithiol Disodium 1,3,4-thiadiazole-2,5-dithiolate
Acute oral toxicity LD50 = 930 mg/kg (males) 9850 mg/kg (males; solution)
2955 mg/kg (males, active ingredient)
Skin irritation Not irritating Not irritating
Eye irritation Eye Dam. Cat. 1 Not irritating
It can be reasonably assumed, that the effects are similar also in ecotoxicity tests.

In summary, the read-across from 1,3,4-Thiadiazole-2,5-dithiol to Disodium 1,3,4-thiadiazole-2,5-dithiolate is justified.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

Version dated 13-Apr-2004

Deviations:

yes

Remarks:

Instead of storage at freezing conditions, t0 specimens were stored at 2-8°C until analysis – this represents a deviation of the study plan which is not considered to have an influence on the integrity of the results and of the test

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

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

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 1.0, 2.0, 4.0, 8.0, 10 mg/L
- Sampling method: Specimens were drawn from freshly prepared treatments and those incubated for 48 hr.
- Sample storage conditions before analysis: The supporting analysis of the specimens could not be performed on the same day of collection of the specimens. Therefore, the specimens were stored at 2-8°C and then analysed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was weighed in ELENDT M4 dilution water followed by stirring on a magnetic stirrer for 10 min (screening test) / 5 min (main test) being necessary to dissolve the test item sufficiently in the test medium. pH of the treatments was not adjusted.
- Controls: ELENDT M4 dilution water without any test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none stated

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: Daphnia magna STRAUS (clone 5)
- Source: obtained from Dr. M. Bergtold, BASF, D-67117 Limburgerhof; the strain originally derived from the Institut National de Recherche Chimique Appliquee, France in 1978
- Feeding during test: During the test the daphnia were not fed.


ACCLIMATION
- Acclimation period: The cultivation of the daphnia is performed in a way that the animals are transferred in new test medium at an interval of 1 to 3 days followed by feeding.
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: Desmodesmus subspicatus- and/or Chlorella-cells
- Feeding frequency: following each transfer in new test medium
- Health during acclimation (any mortality observed): no mortality stated

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Daphnias used for the test are not older than 24 hr. Therefore, in every case, daphnia are transferred into fresh medium one day before starting of the daphnia test in order to get daphnia for the test younger than 24 hr.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

none

Hardness:

250 mg/L CaCO3

Test temperature:

19.3 - 20.6 °C during the definitive main test (20.0 - 20.3 °C during the screening test)

pH:

8.24 - 9.28 during the definitive main test (5.75 - 8.30 during the screening test)

Dissolved oxygen:

8.3 - 8.9 mg O2/L during the definitive main test (8.2 - 8.7 during the screening test)

Nominal and measured concentrations:

Nominal: 0, 1.0, 2.0, 4.0, 8.0, 10 mg/L (definitive main test)
Found (t0): 0.0, 1.05, 2.11, 4.22, 8.37, 10.69 mg/L
Found (t48h): 0.0, 0.99, 1.97, 4.04, 8.16, 10.43 mg/L
Mean (t0 - t48h): 0.0, 1.02, 2.04, 4.13, 8.27, 10.56 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: carboys
- Material, size, headspace, fill volume: glass, 100mL volume
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4 (definitive main test), 2 (screening test)
- No. of vessels per control (replicates): 4 (definitive main test), 2 (screening test)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 (test medium according to OECD 202); the stock solutions with the macronutrients were individually prepared in ultrapure water (for each salt a separate solution). From these individual stock solutions, the medium was prepared.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Photoperiod: 16 hr of light and 8 hr of darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobility, daily

VEHICLE CONTROL PERFORMED: yes, medium only

RANGE-FINDING STUDY
- Test concentrations: 1, 10, 100 mg/L

Reference substance (positive control):

yes

Remarks:

To verify the sensitivity of the test system towards toxic items the effect of potassium dichromate towards different animals of this breed was tested prior to beginning of the test.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

5.95 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

other: EC20

Effect conc.:

4.46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

3.83 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Mortality of control: no immobilisation noted
- Other adverse effects control: none stated
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none stated
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Limit test: no
- Dose-response test: yes
- ECx: EC50 (24 hr) = 0.95 mg/L
- Other: In order to verify the sensitivity of the test system towards toxic items the effect of potassium dichromate towards different animals of this breed was tested prior to beginning of the test. The value of the EC50 (24 hr) in this period of time was at 0.95 mg/L potassium dichromate. This value is in accordance with the recommendations of 0.6 to 2.1 mg/L K2Cr2O7 given in OECD Guideline 202 (Daphnia sp., Acute Immobilisation Test).

Reported statistics and error estimates:

Statistical evaluation was done using the commercial computer program ToxRat Professional Version 3.2.1.

Validity criteria fulfilled:

yes

Conclusions:

The study was performed according to OECD TG 202 without deviations which may have impact on the validity of the study. Thus, the results were so obtained via a scientifically reasonable method. Hence, there is no doubt that the obtained results are not reliable:
Under the conditions used for the test, there was a significant immobilisation of the daphnias at concentrations >2.0 mg/L (definitive main test).
As a conclusion of the analytical part of this study, it can be stated that the concentrations of the test item remained sufficiently stable during incubation. Based on this, the reported effect concentrations should refer to the nominal concentrations tested. The following EC-values were determined accordingly after 48 hours:

Mobility:
LOEC = 4.0 mg/L
NOEC = 2.0 mg/L
EC50 = 5.95 mg/L

Based on these results, the test item does not need to be classified as acute toxic to the aquatic environment. With regard to chronic toxicity, taking into account the facts that the test item is soluble in water, i.e. above the determined EC50 value, and not readily biodegradable, the test item should be classified as aquatic chronic Cat. 2. This refers to the read-across substance as pure solid. The actual registered substance however contains water > 57% water, so the registered substance does only need to be classified as aquatic chronic Cat. 3, out of the following reasons:

The CLP-Regulation 1272/2008 (and amendments) states under Annex I, 4.1.3.4. Classification of mixtures when toxicity data are not available for the complete mixture: bridging principles

4.1.3.4.3. If a mixture is formed by diluting another tested mixture or substance with water or other totally non-toxic material, the toxicity of the mixture can be calculated from the original mixture or substance.

The EC50 is considered relevant here, as only EC50 values are available for determination uf aquatic chronic toxicity. There are two options for interpretation:

Either, water is considered completely inert and only the volume / weight is taken into consideration. With an EC50 value of 5.95 mg/kg of the solid RA-substance, and the corresponding solid DMTD-Na is contained to 43% in the registered aqueous solution, an EC50 value of 13.84 mg/L can be estimated for the solution.

Alternatively, the EC50 value is determined from equation stated under 4.1.3.5.2, assuming a worst case EC50 of 100 mg/L for water:

100% / EC50m = 43% / 5.95 mg/L + 57% / 100 mg/L = 7.80 % / mg/L

EC50m = 100% / 7.80% mg/L = 12.82 mg/L

Either way, an EC50 > 10 mg/L is estimated, and so, being not biodegradable, the registered substance does only need to be classified as aquatic chronic 3.

Executive summary:

2,5-Dimercapto-1,3,4-thiadiazole (“DMTD”) was tested for acute toxicity towards daphnia according to OECD-Test Guideline 202 (version dated 13-Apr-2004) in compliance with GLP. In order to investigate the influence of the test item towards the daphnia, the swimming behaviour of the animals was recorded. Under the conditions used for the test, there was an immobilisation of the daphnia at concentrations >2 mg/L. As a conclusion of the analytical part of this study, it can be stated that the concentrations 2,5-Dimercapto-1,3,4-thiadiazole (“DMTD”) remained sufficiently stable during incubation of 48hr. As a result of the supporting analysis it can be stated that the results of the biological part should be based on the nominal concentrations applied because the recoveries of the nominal concentrations were all >80%.

The following EC-values (effective concentrations) were determined accordingly:

	Basis: Nominal Concentrations [mg/L]
	t24hr	t48hr
NOEC	≥ 10.00	2.00
LOEC	>10.00	4.00
EC10	7.57	3.83
EC20	11.71	4.46
EC50	n.d. (>10.00)	5.95

n.d. = not determined for mathematical reasons.

The test is considered valid as all conditions for validity were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

Version dated 13-Apr-2004

Deviations:

yes

Remarks:

Instead of storage at freezing conditions, t0 specimens were stored at 2-8°C until analysis – this represents a deviation of the study plan which is not considered to have an influence on the integrity of the results and of the test

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,3,4-thiadiazole-2,5-dithiol

EC Number:

214-014-1

EC Name:

1,3,4-thiadiazole-2,5-dithiol

Cas Number:

1072-71-5

Molecular formula:

C2H2N2S3

IUPAC Name:

1,3,4-thiadiazole-2,5-dithiol

Test material form:

solid

Specific details on test material used for the study:

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

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 1.0, 2.0, 4.0, 8.0, 10 mg/L
- Sampling method: Specimens were drawn from freshly prepared treatments and those incubated for 48 hr.
- Sample storage conditions before analysis: The supporting analysis of the specimens could not be performed on the same day of collection of the specimens. Therefore, the specimens were stored at 2-8°C and then analysed.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was weighed in ELENDT M4 dilution water followed by stirring on a magnetic stirrer for 10 min (screening test) / 5 min (main test) being necessary to dissolve the test item sufficiently in the test medium. pH of the treatments was not adjusted.
- Controls: ELENDT M4 dilution water without any test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none stated

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: Daphnia magna STRAUS (clone 5)
- Source: obtained from Dr. M. Bergtold, BASF, D-67117 Limburgerhof; the strain originally derived from the Institut National de Recherche Chimique Appliquee, France in 1978
- Feeding during test: During the test the daphnia were not fed.


ACCLIMATION
- Acclimation period: The cultivation of the daphnia is performed in a way that the animals are transferred in new test medium at an interval of 1 to 3 days followed by feeding.
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: Desmodesmus subspicatus- and/or Chlorella-cells
- Feeding frequency: following each transfer in new test medium
- Health during acclimation (any mortality observed): no mortality stated

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Daphnias used for the test are not older than 24 hr. Therefore, in every case, daphnia are transferred into fresh medium one day before starting of the daphnia test in order to get daphnia for the test younger than 24 hr.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

none

Test conditions

Hardness:

250 mg/L CaCO3

Test temperature:

19.3 - 20.6 °C during the definitive main test (20.0 - 20.3 °C during the screening test)

pH:

8.24 - 9.28 during the definitive main test (5.75 - 8.30 during the screening test)

Dissolved oxygen:

8.3 - 8.9 mg O2/L during the definitive main test (8.2 - 8.7 during the screening test)

Nominal and measured concentrations:

Nominal: 0, 1.0, 2.0, 4.0, 8.0, 10 mg/L (definitive main test)
Found (t0): 0.0, 1.05, 2.11, 4.22, 8.37, 10.69 mg/L
Found (t48h): 0.0, 0.99, 1.97, 4.04, 8.16, 10.43 mg/L
Mean (t0 - t48h): 0.0, 1.02, 2.04, 4.13, 8.27, 10.56 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: carboys
- Material, size, headspace, fill volume: glass, 100mL volume
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4 (definitive main test), 2 (screening test)
- No. of vessels per control (replicates): 4 (definitive main test), 2 (screening test)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 (test medium according to OECD 202); the stock solutions with the macronutrients were individually prepared in ultrapure water (for each salt a separate solution). From these individual stock solutions, the medium was prepared.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Photoperiod: 16 hr of light and 8 hr of darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobility, daily

VEHICLE CONTROL PERFORMED: yes, medium only

RANGE-FINDING STUDY
- Test concentrations: 1, 10, 100 mg/L

Reference substance (positive control):

yes

Remarks:

To verify the sensitivity of the test system towards toxic items the effect of potassium dichromate towards different animals of this breed was tested prior to beginning of the test.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

5.95 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

other: EC20

Effect conc.:

4.46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

3.83 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 5

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Mortality of control: no immobilisation noted
- Other adverse effects control: none stated
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none stated
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Limit test: no
- Dose-response test: yes
- ECx: EC50 (24 hr) = 0.95 mg/L
- Other: In order to verify the sensitivity of the test system towards toxic items the effect of potassium dichromate towards different animals of this breed was tested prior to beginning of the test. The value of the EC50 (24 hr) in this period of time was at 0.95 mg/L potassium dichromate. This value is in accordance with the recommendations of 0.6 to 2.1 mg/L K2Cr2O7 given in OECD Guideline 202 (Daphnia sp., Acute Immobilisation Test).

Reported statistics and error estimates:

Statistical evaluation was done using the commercial computer program ToxRat Professional Version 3.2.1.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The study was performed according to OECD TG 202 without deviations which may have impact on the validity of the study. Thus, the results were so obtained via a scientifically reasonable method. Hence, there is no doubt that the obtained results are not reliable:
Under the conditions used for the test, there was a significant immobilisation of the daphnias at concentrations >2.0 mg/L (definitive main test).
As a conclusion of the analytical part of this study, it can be stated that the concentrations of the test item remained sufficiently stable during incubation. Based on this, the reported effect concentrations should refer to the nominal concentrations tested. The following EC-values were determined accordingly after 48 hours:
Mobility:
LOEC = 4.0 mg/L
NOEC = 2.0 mg/L
EC50 = 5.95 mg/L
Based on these results, the test item does not need to be classified as acute toxic to the aquatic environment. With regard to chronic toxicity, taking into account the facts that the test item is soluble in water, i.e. above the determined EC50 value, and not readily biodegradable, the test item should be classified as aquatic chronic Cat. 2.

Executive summary:

2,5-Dimercapto-1,3,4-thiadiazole (“DMTD”) was tested for acute toxicity towards daphnia according to OECD-Test Guideline 202 (version dated 13-Apr-2004) in compliance with GLP. In order to investigate the influence of the test item towards the daphnia, the swimming behaviour of the animals was recorded. Under the conditions used for the test, there was an immobilisation of the daphnia at concentrations >2 mg/L. As a conclusion of the analytical part of this study, it can be stated that the concentrations 2,5-Dimercapto-1,3,4-thiadiazole (“DMTD”) remained sufficiently stable during incubation of 48hr. As a result of the supporting analysis it can be stated that the results of the biological part should be based on the nominal concentrations applied because the recoveries of the nominal concentrations were all >80%.

The following EC-values (effective concentrations) were determined accordingly:

	Basis: Nominal Concentrations [mg/L]
	t24hr	t48hr
NOEC	≥ 10.00	2.00
LOEC	>10.00	4.00
EC10	7.57	3.83
EC20	11.71	4.46
EC50	n.d. (>10.00)	5.95

n.d. = not determined for mathematical reasons.

The test is considered valid as all conditions for validity were met.