1,4,4-trimethyl-2,3-diazabicyclo[3.2.2]non-2-ene 2,3-dioxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

TAOBN did not cause a positive mutagenic response in either the presence or absence of Arclor-inducted rat liver S9. While there was no independent repeat, the supporting study also concluded TAOBN was not mutagenic in the microbial assays either in the presence or absence of a liver microsomal system.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Haskel No. 9864-02

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain / cell type:

S. typhimurium TA 98

Metabolic activation:

with and without

Metabolic activation system:

rat-liver homogenate activation system (S9)

Test concentrations with justification for top dose:

With activation: 10, 12, 24, 36, 48, 60 µg/plate
Without activation: 2, 4, 6, 8, 10 µg/plate

Vehicle / solvent:

Distilled water

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Positive control for assay in the presence of activation

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Positive control for assay in the absence of activation

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Positive control for assay in the absence of activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Negative control for assay in the absence of activation

Untreated negative controls:

yes

Remarks:

S9 negative control included in the activated assay to reassure any activity of the compound in the absence of the S9 activator mixture

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- All plates were incubated at 37 °C for 48 hours.

NUMBER OF REPLICATIONS: 2 plates

Statistics:

Average number of revertants from two plates

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

The test item was tested in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100. The test item was not mutagenic in the microbial assays either in the presence or absence of a liver microsomal system.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Haskell Number 26185
- Purity: 99.90%

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

The dose levels tested were 3.3, 10, 33, 100, 333, and 1000 µg per plate with E. coli, TA100 in the presence of S9 activation and with TA1535 in the absence of S9 activation and 1.0, 3.3, 10, 33, 100 and 333 µg per plate with all remaining conditions. In the repeat test, the dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate.

A preliminary toxicity test was used to establish the dose-range over which the test substance would be tested.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 100% ethanol
- Justification for choice of solvent/vehicle: selected based on the solubility of the test substance and compatibility with the target cells. Other solvents tested included dimethyl sulfoxide (DMSO) and acetone.

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Positive control for WP2 uvrA strain (for concentrations of 1.0 µg/plate and 1000 µg/plate)

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Positive control for TA98 strain

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Positive control for TA100 and TA1535 strains

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Positive control for TA1537 strain

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Positive control for WP2 uvrA (for concentration of 1000 µg/plate)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- in agar (plate incorporation)

DURATION:
- Preincubation period: overnight
- Exposure duration: Not applicable. Cultures harvested via spectrophotometric monitoring of culture turbidity rather than by duration of incubation since overgrowth of cultures can cause loss of sensitivity of some mutagens
- Expression time: Cultures harvested in late log phase
- Fixation time: Each culture was monitoring spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of approximately 109 cells/mL.

NUMBER OF REPLICATIONS: 3 plates/test concentration

Evaluation criteria:

Revertant colonies, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity.

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported. For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100, and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2.0-times the mean vehicle control value.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitate was observed

RANGE-FINDING/SCREENING STUDIES:
- dose levels tested: 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333, 5000 µg per plate
- no precipitate observed
- toxicity observed at 67, 100, 333, or 667 µg per plate
- based on findings, max doses plated in mutagenicity test were 1000 µg per plate with E. coli, TA100 in the presence of S9 activation and with TA1535 in the absence of S9 activation and 333 µg per plate with all remaining conditions.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Strain TA98:
Without activation:
Mean: 239 revertants/plate
SD: 158
Range: 30 - 1581 revertants/plate

With activation:
Mean: 679 revertants/plate
SD: 383
Range: 40 - 2294 revertants/plate

Strain TA100:
Without activation:
Mean: 572 revertants/plate
SD: 149
Range: 240 - 1945 revertants/plate

With activation:
Mean: 904 revertants/plate
SD: 437
Range: 163 – 2922 revertants/plate

Strain TA1535:
Without activation:
Mean: 319 revertants/plate
SD: 131
Range: 16 - 978 revertants/plate

With activation:
Mean: 137 revertants/plate
SD: 80
Range: 11 – 2246 revertants/plate

Strain TA1537:
Without activation:
Mean: 634 revertants/plate
SD: 375
Range: 13 - 2389 revertants/plate

With activation:
Mean: 127 revertants/plate
SD: 133
Range: 12 – 2060 revertants/plate

Strain WP2 uvrA:
Without activation:
Mean: 164 revertants/plate
SD: 147
Range: 14 - 1809 revertants/plate

With activation:
Mean: 390 revertants/plate
SD: 255
Range: 22 – 1493 revertants/plate



- Negative (solvent/vehicle) historical control data:
Strain TA98:
Without activation:
Mean: 16 revertants/plate
SD: 6
Range: 5 - 59 revertants/plate

With activation:
Mean: 21 revertants/plate
SD: 7
Range: 5 - 58 revertants/plate

Strain TA100:
Without activation:
Mean: 148 revertants/plate
SD: 36
Range: 68 - 262 revertants/plate

With activation:
Mean: 152 revertants/plate
SD: 38
Range: 74 - 271 revertants/plate

Strain TA1535:
Without activation:
Mean: 15 revertants/plate
SD: 6
Range: 3 - 46 revertants/plate

With activation:
Mean: 13 revertants/plate
SD: 5
Range: 2 – 50 revertants/plate

Strain TA1537:
Without activation:
Mean: 7 revertants/plate
SD: 3
Range: 1 - 22 revertants/plate

With activation:
Mean: 8 revertants/plate
SD: 3
Range: 1 – 28 revertants/plate

Strain WP2 uvrA:
Without activation:
Mean: 14 revertants/plate
SD: 4
Range: 5 - 52 revertants/plate

With activation:
Mean: 14 revertants/plate
SD: 4
Range: 4 – 52 revertants/plate

In the mutagenicity test, no positive mutagenic response was observed. The dose levels tested were 3.3, 10, 33, 100, 333, and 100 µg per plate with E. coli, TA100 in the presence of S9 activation and with TA1535 in the absence of S9 activation and 1.0, 3.3, 10, 33, 100 and 333 µg per plate with all remaining conditions. In the repeat test, the dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate. No precipitate was observed. Toxicity was observed beginning at 100, 150, 333 or at 1000 µg per plate.

Conclusions:

TAOBN did not cause a positive mutagenic response in either the presence or absence of Arclor-inducted rat liver S9. The study was concluded to be negative without conducting a confirmatory (independent repeat) test because no unique metabolism requirements were known about the test substance and because no equivocal responses were observed in the test that would suggest further testing is warranted.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

As two independent AMES studies were negative for gene mutation, the substance does not meet the criteria for CLP (EC 1272/2008 as amended) classification for germ cell mutagenicity.