N,N'-ethylenebis(3,4,5,6-tetrabromophthalimide)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was negative (non mutagenic) in three standard in vitro assays, the bacterial gene mutation assay, a gene mutation assay in mammalian cells and a chromosomal aberration assay in mammalian cells, bit with and without metabolic activation.

Link to relevant study records

Reference

Reference 1

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:

1982

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed by an experienced laboratory using an accepted methodology that included the typical Salmonella strains plus E. coli, both with and without metabolic activation.

Qualifier:

according to guideline

Guideline:

other: Japanese Industrial Safety and Health Law, 1979,

Qualifier:

equivalent or similar to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Typical for the bacterial strains tested.

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: tryptophan-deficient E.coli WP2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9

Test concentrations with justification for top dose:

10, 50, 100, 500, 1000, 5000 ug/plate; duplicate plates/dose, bacterial strain, and activation

Vehicle / solvent:

DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Remarks:

2 -(2 -furyl)-3 -(5 -nitro-2 -furyl)acrylamide, N-ethyl-N'nitro-N-nitrosoguanidine, 9 -Aminoacridine, 2 -nitrofluorene, 2 -aminoanthracene

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

See attached document for table of results.

Conclusions:

Interpretation of results (migrated information):
negative

EBTBP was not mutagenic to 5 strains of Salmonella typhimurium and E. coli WP2 uvrA when tested with and without S9 metabolic activation.

Executive summary:

In the Ames assay, the tester strains used wereSalmonellaTA98, TA100, TA1535, TA1537, and TA1538 andE. coli, WP2 uvrA. Each strain was tested with and without a source of exogenous metabolic activation of Arochlor-induced rat liver microsomes. EBTBP dose levels were 0, 1, 10, 100, 500, 1,000 and 5,000 ug/plate. Positive and negative controls were included, and performed as expected. No increase in revertant colonies was found at any EBTBP dose level either in the presence or absence of microsomal enzymes. EBTBP was not genetically active in this assay.

Reference 2

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:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Performed by experienced laboratory under Good Laboratory Practices and established internattional guidelines using the commercial product as test material.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Arochlor-induced rat hepatic S-9

Test concentrations with justification for top dose:

Non-activated, 4 hr treatment, 16 hr recovery: 5, 10, 25, 50, 100 ug/mL
Non-activated, 20 hr treatment, 0 hr recovery:5, 10, 25, 50, 100, 250, 350, 500, 700 ug/mL
S9-activated, 4 hr treatment, 16 hr recovery: 5, 10, 25, 50, 100 ug/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

Positive controls:

yes

Remarks:

see attached Summary

Positive control substance:

mitomycin C

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

preliminary study

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

See Tables of Results under Attached Document.

Conclusions:

Interpretation of results (migrated information):
negative

EBTBP did not induce chromosome aberrations in Chinese hampster ovary cells when tested with or without metabolic activation.

Executive summary:

See attached EBTBP Abb Summary.pdf

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19.10.2016 to 8.5.2017

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)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro gene mutation in mammalian cells

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Albemarle Corporation / W95276A
- Expiration date of the lot/batch: 13 September 2018
- Purity test date: >= 99.9 %, Nov 2, 2016


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle:stable suspension in DMSO
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: none

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: The maximum concentration in a suspension suitable for dosing was 200 mg/mL. This stoc formulation was used at 1% to give the maximum recommended concnetration level of 2000 micro-g/mL

Target gene:

HPRT locus

Test concentrations with justification for top dose:

Preliminary test: 0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000, 2000 mico-g/mL.Precipitation at the end of th eexposure period was observed without metabolic activation at and above 31.25 micro-g/mL, and with metabolic activation at and above 62.5 micro-g/mL. No indications of any marked concentration related reductions in clonign efficiency were observed. The maximum concentration for th emain experiment was selected based on the onset of precipitation of the test item, i.e. 32 micro-g/mL without metabolic activation and 62.5 micro-g/mL with metabolic acitvation.

Vehicle / solvent:

DMSO. No suitable vehicle where the test item coudl be dissolved was found in preliminary experiments.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

EMS Ethyl methane sulphonate without S9, Dimethyl benzanthracene with S9

Positive control substance:

9,10-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension in serum free minimal essential medium
- Cell density at seeding (if applicable):1x10E7 cells/225 cm2 flask yielding at least 20 x 10E6 viable cells pe flask for dosing

Metabolic activation: Phenobarbitone/beta-Napthoflavone rat liver S9-mix

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 day (subcultured and maintained on days 2 to 5)
- Selection time (if incubation with a selection agent): 7 days with 6-thioguanidine
- Fixation with methanol (5 min) and staining with Giemsa after the selection time

SELECTION AGENT (mutation assays): 6 thioguanidine

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Fixation of petri dishes by aspirating off media, wahsing with phosphate buffered saline and fixing for 5 min with methanol. Staining with 10% Giemsa solution for 5 min.

NUMBER OF CELLS EVALUATED: 2x10E5 cells per petri dish, 10 replicates

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency: triplicates of 200 cells/25 cm2 flask in 5 mL of MEM with 10% FBS. Incubayion for 6 to 7 days, fixation with methanol and stain with Giemsa as above. Colonies were manually counted, counts recorded for each culture and the percentage clonign efficiency calculated for each dose group.

Rationale for test conditions:

all according to OECD 476 guideline

Evaluation criteria:

Validity Criteria:
- The average absolute cloning efficiency of the Day 7 negative controls should exceed 50%.
- The background (spontaneous) mutant frequency of the vehicle controls is generally within the historical range. The background values for the with and
without-activation segments of a test may vary even though the same stock populations of cells may be used for concurrent assays.
- The concurrent positive controls should induce responses that are comparable with those generated in the historical positive control range and produce a
statistically significant increase compared with the concurrent negative control.
-The criteria for selection of the maximum concentration have been met. Test items with little or no mutagenic activity, should include an acceptable assay
where concentrations of the test item have reduced the clonal survival to approximately 10 to 15% of the average of the negative controls, reached the
maximum recommended concentration, or include the lowest precipitating concentration. Where a test item is excessively toxic, with a steep response curve, a concentration that is at least 75% of the toxic concentration should be used.Treatments that reduce relative clonal survival to less than ten percent will not be scored for mutant frequency in the assay.
-Adequate numbers of cells and concentrations are analyzable. Mutant frequencies are normally derived from sets of ten dishes/flasks for the mutant colony count and three dishes for cloning colony counts. To allow for contamination losses it is acceptable to score a minimum of eight mutant selection dishes and two cloning efficiency flasks.
- Five concentrations of test item, in duplicate, in each assay will normally be assessed for mutant frequency. A minimum of four analyzed duplicate concentrations is considered necessary in order to accept a single assay for evaluation of the test item.

Statistics:

No statsitics if no indicationof any increase of mutant frequency. Otehrwise comparison with vehicle control by Student's t-test.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

At the end of the exposure period, the onset of precipitate of the test item was observed at 32 µg/ml in the absence of metabolic activation, and at 32 µg/mL in the presence of metabolic activation. Therefore, following the recommendations of the OECD 490 guideline, the 64 µg/ml dose level in the presence of metabolic activation was excluded from the analysis as it was considered to be surplus to requirements.
The Day 0 and Day 7 cloning efficiencies for the exposure groups in the absence and presence of metabolic activation are presented in Tables 2 and 3. There were no concentration-related reductions in the Day 0 cloning efficiency values in either the absence or presence of metabolic activation. There was also no evidence of any reductions in the Day 7 cloning efficiencies in any of the concentration levels, therefore indicating that residual toxicity had not occurred.
The mutation frequency counts and mean mutation frequency per survivor values are presented in Table 2 and Table 3. The test item did not induce
any toxicologically significant or concentration-related increases in the mutant frequency at any of the concentration levels in the main test, in either the absence or presence of metabolic activation.
The vehicle control values were all considered to be within an acceptable range, and the positive controls gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.

Conclusions:

1,2-Bis-(tetrabromophthalimido)ethane was shown to be non-mutagenic in the HPRT Test with Chinese hamster V79 cells, according to OECD TG 476 and GLP, both with and without metabolic activation at test concentrations below the onset of precipitation as specified in the guideline. Positve and solvent controls showed the expected response in the test system.

Executive summary:

Chinese hamster (V79) cells were treated with 1,2-Bis-(tetrabromophthalimido)ethane at up to eight concentrations, in duplicate, together with vehicle (DMSO) and positive controls in both the absence and presence of metabolic activation.   The concentrations used in the main test were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by the onset of test item precipitate in both the absence and presence of metabolic activation, as recommended by the OECD 476 guidelines.  The concentrations of test item plated for cloning efficiency and expression of mutant colonies were as follows:

4-hour without S9: 1, 2, 4, 8, 16, 32 micro-g/mL

4-hour with S9 (2%): 2, 4, 8, 16, 32, 64 micro-g/mL

The vehicle (DMSO) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus.

The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant or concentration-related increases in mutant frequency at any of the concentration levels in the main test, in either the absence or presence of metabolic activation.

Therfore it can be concluded that 1,2-Bis-(tetrabromophthalimido)ethane was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The substance was negative in a bacterial genotoxicity test, a mammalian gene mutation test and a mammalian cell chromosomal aberration study. Therefore it can be concluded that from the consistent results of the in vitro assays there is no indication for a genotoxic potential of the substance.

Short description of key information:
The substance was negative in a bacterial genotoxicity test, a mammalian cell gene mutation assay and a mammalian cell chromosomal aberration study, both in presence and absence of a metabolic activation system. All studies were performed in accordance with internationally accepted test guidelines and are considered relaible without restrictions.

Therefore it can be concluded that from the consistent results of the in vitro assays there is no indication for a genotoxic potential of the substance.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No indication of a possible genotoxic effect was observed in in vitro genotoxicity studies and repeated dose studies did not reveal any substance related effect. Therefore a classification for gentoxicity is not warranted.