2-(butylnitroamino)ethyl nitrate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9/20/91 - 12/31/91

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

2-(butylnitroamino)ethyl nitrate

EC Number:

279-976-7

EC Name:

2-(butylnitroamino)ethyl nitrate

Cas Number:

82486-82-6

Molecular formula:

C6H13N3O5

IUPAC Name:

butyl(nitro)[2-(nitrooxy)ethyl]amine

Method

Target gene:

selected histidine loci

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Source: Dr. Bruce N. Aries
University of California
Biochemistry Dept.
Berkeley, California 94720

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

s9 fraction

Test concentrations with justification for top dose:

Butyl-nena 167, 500, 1670, 5000, 7500 and 10,000 µg/plate with S9, and 50.0, 167, 500, 1670, 3330 and 5000µg/plate without S9.
BuNENA was re-evaluated in a confirmatory assay at doses of 16.7, 50.0, 167, 500, 1670 and 5000µg/plate with and without S9 (doses evaluated in the confirmatory assay were adjusted based upon toxicity observations made in the original assay).

Vehicle / solvent:

molten top agar

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-anthramine

Details on test system and experimental conditions:

Treatment for the mutation assay was performed exactly as described in the toxicity prescreen, except that the test and control articles were evaluated in triplicate cultures in all five tester strains in the presence and absence of an exogenous metabolic activation system (S9). Cultures treated in the presence of S9 contained 0.5 ml of the S9 mixture. The S9 mixture contained 8mM MgCl2, 33mM KCl, 4mM NADP, 5mM gluccse-6-phosphate, lOOmM Na2HP04 (pH 7.4) and 6% (v/v) Aroclor 1254-induced male Sprague-Dawley rat liver homogenate.

Rationale for test conditions:

based upon preliminary toxicity assays

Evaluation criteria:

A positive result is defined as a statistically significant, dose-dependent increase in the number of histidine-independent revertants with at least one dose level inducing a revertant frequency that is two-fold the spontaneous solvent control value. Statistical analyses were performed using the program developed by Snee and Irr (1981), with significance established at the 95% confidence limit. If the test article does not induce a statistically significant, dose-dependent increase in revertant
frequency, but does induce a revertant frequency at one dose level that is two-fold the spontaneous control value, the result is considered equivocal. A negative result is defined as the absence of a statistically significant or dose-dependent increase in the number of histidine-independent revertants.

Statistics:

Statistical analyses generally are performed only when a 50% increase in revertant frequency, relative to the concurrent negative controls, is observed. This 50% "trigger" was selected based upon the normal, spontaneous variation observed among replicate negative control cultures (see above), as well as spontaneous fluctuation observed in this laboratory among groups of cultures treated with a variety of test article judged to be negative in this assay. However, statistical analyses also were performed in a limited number of cases where smaller (but apparently dose-dependent) increases were observed, or where such 50% increases were observed in the original, but not the confirmatory, mutation assay. Comparisons of mutagenic potential of the five test articles were made using the uniformly positive results observed in strain TA1535 with S9. The slopes of the dose-response curves were recalculated for the initial linear portion of the response (by simple linear regression on the untransformed data), corrected for molecular weight, and used to rank-order the five test articles.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Any other information on results incl. tables

Results of the toxicity prescreen indicated n-butyl-2 -nitratoethyl nitramine (BuNENA) was not toxic to either strain at doses of 50.0, 167 and 500 pq/plate. Inhibited growth was observed in both tester strains at doses of 1670 and 5000 µg/plate. In addition, the test article was freely soluble at all doses evaluated. Based upon these findings, BuNENA was evaluated in the mutation assay in all five tester strains at doses of 167, 500, 1670, 5000, 7500 and 10,000 µg/plate with S9, and 50.0, 167, 500, 1670, 3330 and 5000 µg/plate without S9 (page 25). Inhibited growth was again observed in all tester strains at doses of 500, 1670, 5000, 7500 and/or 10,000 µg/plate with S9, and at doses of 1670, 3330 and/or 5000 µg/plate without S9. The test article again was freely soluble at all doses evaluated. Revertant frequencies for all doses of BuNENA in strains TA1537, TA1538, TA98 and TA100 with and without S9 approximated or were less than, those observed in the concurrent negative control cultures. In contrast, statistically significant, dose-dependent increases in revertant frequencies, to approximately 6.8- to 1.6- fold control values, were observed in strain TA1535 with and without S9, respectively. BuNENA was re-evaluated in a confirmatory assay at doses of 16.7, 50.0, 167, 500, 1670 and 5000 µg/plate with and without S9 (doses evaluated in the confirmatory assay were adjusted based upon toxicity observations made in the original assay). Inhibited growth was again observed in all tester strains at doses of 500, 1670 and/or 5000 µg/plate with and/or without S9. Revertant frequencies for all doses of MeNENA in strains TA1537, TA1538, TA98 and TA100 with and without S9, and in strain TA1535 without S9, approximated or were less than control values. Statistically significant, dosedependent increases in revertant frequencies, to approximately 3.5-fold control values, were observed in strain TA1535 with S9.

Applicant's summary and conclusion

Conclusions:

All five test articles reproducibly induced statistically significant, dose-dependent increases in revertant frequencies, to approximately 2.6- to 20-fold control values, in tester strain TA1535 with S9. Statistically significant, dose-dependent increases in revertant frequencies, to approximately 1.6- to 10- fold control values, also were reproducibly observed for MeNENA, EtNENA, and DNPA/F ±DPA in strain TA1535 without S9. In addition, statistically significant and/or dose-dependent increases in revertant frequencies, to approximately 1.3- to 3.1- fold control values, also were reproducibly observed in strain TA100 for DNPA/F ±DPA with and without S9, and for EtNENA with S9. Similar, but unconfirmed, increases in revertant frequencies, to approximately 1.4- to 1.8-fold control values were observed for MeNENA (TA100 +S9), EtNENA (TA98 -S9), BuNENA (TA1535 -S9), DNPA/F +DPA (TA98 -S9), and DNPA/F -DPA (TA98 +S9). These latter unconfirmed increases, however, are considered to be statistical aberrations due to random fluctuation of the spontaneous revertant frequencies. All positive and negative control values in all assays were within acceptable limits. Therefore, the results for all five test articles were positive in the Ames/Salmonella Plate Incorporation Assay under the conditions, and according to the criteria, of the test protocol. On the basis of the results observed in strain TA1535 with S9, the rank order of mutagenic potential (revertants per ^mol/plate) is EtNENA > BuNENA > DNPA/F +DPA = DNPA/F -DPA » MeNENA.