Diammonium 2,2'-dithiodiacetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance did not induce gene mutation or chromosome abberation in studies according to OECD 471, 473 and 476

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, GLP incl. certificate

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

acc. 440/2008 B.10

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Metabolic activation:

with and without

Test concentrations with justification for top dose:

29.1, 50.9, 89.1, 156.0, 273.0, 477.7, 835.9, 1462.9, 2560.0, 4480.0 µg/ml active ingredient

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Positive controls:

yes

Positive control substance:

cyclophosphamide

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results: negative

It can be stated, that under experimental conditions reported, the test material did not induece structural cromosomal aberrations in human lymphocytes in vitro

Therefore diammonium dithiodiglycolate is considered to be non-clastogenic in this chromosome aberration test, when tested up to the highest required concentration.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine reversion

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (from livers of male Sprague-Dawley rats  treated byphenobarbitone/beta-naphtoflavone 80/10mg per day)

Test concentrations with justification for top dose:

0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Two distinct experiments were performed using these doses.

Vehicle / solvent:

sterile distilled water

Details on test system and experimental conditions:

METHOD DETAILS:
- Test concentrations:
. Preliminary study (on TA100): 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
. Main study: see the "Test concentration" field
- Number of replicates: 2
- Positive controls:
without S9-mix
. for TA1535 and TA100: N-ethyl-N'-nitro-N-nitrosoguanidine (5 and 2  µg/plate respectively)
. for TA1537: 9-aminoacridine (80 µg/plate)
. for TA98: 4-Nitroquinoline-1-oxide (0.2 µg/plate)
. for TA102: mitomycin C (0.5 µg/plate)
with S9-mix
. for TA1535, TA1357 and TA100: 2-aminoanthracene (2; 2 and 1µg/plate  respectively)
. for TA98: benzo(a)pyrene (5 µg/plate)
. for TA102: 1,8-Dihydroxyanthraquinone (10 µg/plate)
- Pre-incubation time: no
- Pre-incubation temperature: no
- Incubation time: 48h
- Incubation temperature: 37°C

EXAMINATION:
- Bacterial toxicity: determined by examination of background lawn growth
- Number of revertants / plate

ANALYTICAL DEVICE:  Colonies were counted electronically using a Domino Colony Counter.

Evaluation criteria:

The reverse mutation assay may be considered valid if the following  criteria are met: All tester strain cultures exhibit a characteristic number of spontaneous  revertants per plate in the vehicle and untreated controls. Acceptable  ranges are presented below with historical control ranges for 2001 and  2002 presented in attached Table.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

The test material was considered to be NON MUTAGENIC under the conditions of this test.

Executive summary:

Due to the result of the test the material has not to be classified.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Dept. of Environment, Hessia, Germany

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

The maximum concentration of the test item was 4480 µg/mL (equal to approximately 10 mM of the active ingredient) during 4 hours treatment with and without metabolic activation. Six to seven concentrations were tested with and without metabolic activation.

Vehicle / solvent:

deionised water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION
in medium
DURATION
- Preincubation period: The cells are subcultured two times prior to treatment.
- Exposure duration: In the first experiment the treatment duration was 4 hours with and without metabolic activation. In the second experiment the cells were exposed to the test item for 4 hours with and for 24 hours without metabolic activation.

SELECTION AGENT (mutation assays)
5 µg / mL TFT (Trifluorothymidine)

DETERMINATION OF CYTOTOXICITY
- Method: The viability (cloning efficiency) was determined by seeding about 2 cells per well into microtiter plates (same medium without TFT). The plates were incubated at 37°± 1.5 °C in 4.5 % CO2/95.5 % water saturated air for 10 - 15 days. Then the plates were evaluated.

Evaluation criteria:

- A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10E6 cells above the corresponding solvent control.
- Results of test groups are generally rejected if the relative total growth is less than 10 % of the vehicle control.
- In accordance with their size the colonies were classified into two groups. Criteria to determine colony size were the absolute size of the colony (more than 1/3 of a well for large colonies) and the optical density of the colonies (the optical density of the small colonies is generally higher than the optical density of the large ones).

Statistics:

- A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software.
- The number of mutant colonies of the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No precipitation of the test item visible to the naked eye was noted in any experimental part up to the maximum concentration.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

The substance DADTDG did not induce mutations in the mouse lymphoma thymidine kinase locus assay at acceptable levels of cytotoxicity using the cell line L5178Y in the absence and presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

All three in vitro tests in genetic toxicity showed negative results. Therefore, there is no need to carry out in vivo studies in genetic toxicity. There is no reason to believe that the negative results would not be relevant to humans.

Justification for selection of genetic toxicity endpoint
Conclusion based on the following assays: Bacterial reverse mutation assay (Ames test); Mammalian cell gene mutation assay; In vitro mammalian chromosome aberration test

Short description of key information:
Negative in all tests conducted:
- Ames test with S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 102 (met. act.: with and without) (OECD TG 471 and GLP); no toxicity was observed up to a concentration of 5000 µg/plate.
- Mammalian cell gene mutation assay with Chinese hamster lung fibroblasts (V79) (met. act.: with and without) (OECD Guideline 476 and GLP); cytotoxicity: preliminary toxicity tests
- In vitro mammalian chromosome aberration test with Chinese Hamster Lung (CHL) cells (met. act.: with and without) (OECD Guideline 473 and GLP); cytotoxicity: no

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data, no classification is needed.