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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 September 1991 - 31 October 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
E. coli WP2 or S. typhimurium TA102 were not tested.
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-(methylnitroamino)ethyl nitrate
EC Number:
241-168-7
EC Name:
2-(methylnitroamino)ethyl nitrate
Cas Number:
17096-47-8
Molecular formula:
C3H7N3O5
IUPAC Name:
methyl(nitro)[2-(nitrooxy)ethyl]amine
Test material form:
solid
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
Samples of MeNENA were uncapped and placed in a desiccator (with desiccant) for approx. 24 hours prior to use, to remove the added water.

Method

Target gene:
Histidine-requiring gene in Salmonella typhimurium.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : male sprague-Dawley rat
- method of preparation of S9 mix: The S9 mixture contained 8mM MgCI2 , 33mM KCl, 4mM NADP, 5mMglucose-6-phosphate, 100mM Na2 HP04 (pH 7.4) and 6% (v/v) Aroclor 1254 induced liver homogenate .
- concentration or volume of S9 mix and S9 in the final culture medium: Cultures treated in the presence of S9 contained 0.5 mL of the S9 mixture.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): S9 mix was evaluated for sterility.
Test concentrations with justification for top dose:
167, 500, 1670, 5000, 7500 and 10000 μg/plate.
In a preliminary toxicity test performed with strains TA1538 and TA100 (-S9), no cytotoxicity was found at doses up to 5000 μg/plate. In addition, the test article was freely soluble at all doses evaluated.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO), Lot #902873, supplied by Fisher Scientific (Fairlawn, NJ).
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA100/-S9 and TA1535/-S9 (10 μg/plate)
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537/-S9 (150 μg/plate)
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA98/-S9 and TA1538/-S9 (5 μg/plate)
Positive controls:
yes
Positive control substance:
other: 2-anthramine
Remarks:
TA 1535, TA 1537, TA 98 and TA 100 and 1538 / +S9 (2.5 μg/plate)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : triplicate
- Number of independent experiments : one initial experiment and one confirmatory assay (in case of positive results in the first assay)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1-2 x 109 cells/mL.
- Test substance added in agar (plate incorporation). Without metabolic activation, 0.1 ml tester strain, 0.1 ml of the appropriate concentration of the test article or solvent and 2 ml of molten top agar (supplemented with 0.5mM histidine/0.5mM biotin) were mixed. For the assay with metabolic activation, 0.5 ml of the S9 mixture was also added.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition.
- Any supplementary information relevant to cytotoxicity: Toxicity of the substance was first evaluated in a prescreen by treating duplicate cultures of strains TA1538 and TA100 at doses of 0.0 (solvent control), 50.0, 167, 500, 1670 and 5000 μg /plate in the absence of S9. The toxicity was determined by evaluating the growth of the background lawn and/or frequency of spontaneous revertants.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Following incubation for 48 hours, revertant colonies were enumerated on an Artek electronic colony counter interfaced with an IBM PC/AT computer for data acquisition.
Solvent and positive controls were scored first, and test article treated cultures were scored only if the average negative control values were within historical ranges (x ± 2SD;see table below).

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

Results and discussion

Test resultsopen allclose all
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:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
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:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
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:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES (if applicable):
Results of the toxicity prescreen indicated MeNENA was not toxic (characterized as normal background lawn growth) to strains TA1538 and TA100 at doses of 50.0, 167, 500, 1670 and 5000 µg/plate in the absence of S9. In addition, the test article was freely soluble at all doses evaluated.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : see table below.

For all test methods and criteria for data analysis and interpretation:
- First assay: statistically significant, dose-dependent increases in revertant frequencies were observed in strain TA1535 with (2.6-fold) and without S9 (1.6-fold).
-Confirmatory assay: statistically significant, dose-dependent increases in revertant frequencies were observed in strains TA1535 (13-fold) and TA100 (1.4-fold) with S9, and in strain TA1535 without S9 (2.0-fold).

Ames test:
- Signs of toxicity: see table below
- Mean number of revertant colonies per plate and standard deviation: see table below.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Not reported
- Negative (solvent/vehicle) historical control data: see table below.

Any other information on results incl. tables

Table 1. Historical Data - Spontaneous Revertants*

Strain S9 n

Average

(± 1SD)

Range

(X± 2SD)

TA1535 - 173 9.65 ± 2.78 4.08 - 15.2
+ 176 10.3 ± 2.80 4.67 - 15.9
TA1537 - 172 7.87 ± 2.54 2.79 - 12.9
+ 171 9.50 ± 2.75 4.00 - 15.0
TA1538 - 177 5.92 ± 2.41 l.11 - 10.7
+ 178 13.5 ± 3.60 6.26 - 20.6
TA98 - 186 19.0 ± 4.92 9.14 - 28.8
+ 195 27.4 ± 6.77 13.9 - 40.9
TA100 - 184 83.8 ± 15.9 52.0 - 116
+ 188 96.9 ± 16.0 64.8 - 129

*January 1, 1990 - September 30, 1991

Table 2. Original Mutation Assay on Me-NENA

CONTROLS
  AVERAGE REVERTANTS/PLATE
Solvent controls   S9 TA1535 TA1537 TA1538 TA98 TA100
DMSO (100µL)   (-) 15 (4) 10 (2) 3 (3) 21 (6) 100 (9)
DMSO (100µL)   (+) 16 (2) 10 (3) 14 (2) 27 (1) 107 (18)
               
Positive controls (µg/plate)            
SODIUM AZIDE 10.0 (-) 1356 * (96) ---(---) ---(---) ---(---) 1312 * (114)
9-AMINOACRIDINE 150 (-) ---(---) 1279 * (38) ---(---) ---(---) ---(---)
2-NITROFLUORENE 5.00 (+) ---(---) ---(---) 471 * (66) 408 * (44) ---(---)
2-ANTHRAMINE 2.50 (+) 117 *(24) 604 * (166) 1671 * (133) 2298 * (212) 2370 * (195)
               
TEST ARTICLE: Me-NENA
DOSE LEVEL (µg/plate) S9 TA1535 TA1537 TA1538 TA98 TA100
167 (-) 14 (6) 9 (0) 5 (3) 18 (1) 85 (17)
500 (-) 17 (4) 8 (2) 3 (2) 17 (7) 100 (22)
1670 (-) 17 (5) 8 (2) 4 (2) 17 (3) 102 (24)
5000 (-) 21 (3) 10 (5) 4 (3) 20 (7) 100 (11)
7500 (-) 20 (5) 5 (1) 4 (1) 18 (3) 88 (22)
10000 (-) 24 (3) 7 (1) 3 (2) 18 (2) 99 (9)
               
167 (+) 17 (2) 12 (6) 14 (5) 30 (6) 103 (13)
500 (+) 21 (8) 14 (9) 13 (2) 22 (5) 121 (10)
1670 (+) 32 * (9) 9 (3) 11 (6) 27 (2) 107 (15)
5000 (+) 38 * (9) 5 (2) 14 (3) 23 (6) 121 (7)
7500 (+) 41 * (3) 8 (4) 14 (2) 26 (4) 122 (25)
10000 (+) 37 * (11) 6 (6) 8 (4) 24 (4) 97 (12)

Data reported as: Mean (Standard Deviation).

*Positive response (≥ 2X solvent control value. Only two-fold or greater increases are indicated)

Apparently normal growth all strains/doses +/- S9.

No precipitate.

Table 3. Confirmatory Mutation Assay on Me-NENA

CONTROLS
  AVERAGE REVERTANTS/PLATE
Solvent controls   S9 TA1535 TA1537 TA1538 TA98 TA100
DMSO (100µL)   (-) 11 (4) 9 (7) 4 (3) 19 (2) 114 (3)
DMSO (100µL)   (+) 9 (4) 7 (1) 12 (5) 24 (3) 131 (12)
               
Positive controls (µg/plate)            
SODIUM AZIDE 10.0 (-) 1140 * (62) ---(---) ---(---) ---(---) 926 * (133)
9-AMINOACRIDINE 150 (-) ---(---) 1231 * (55) ---(---) ---(---) ---(---)
2-NITROFLUORENE 5.00 (+) ---(---) ---(---) 273 * (26) 287 * (39) ---(---)
2-ANTHRAMINE 2.50 (+) 137 *(22) 603 * (63) 1507 * (130) 2173 * (128) 2055 * (239)
               
TEST ARTICLE: Me-NENA
DOSE LEVEL (µg/plate) S9 TA1535 TA1537 TA1538 TA98 TA100
167 (-) 8 (4) 4 (2) 3 (2) 18 (3) 113 (24)
500 (-) 9 (4) 3 (2) 1 (0) 11 (4) 110(22)
1670 (-) 10 (4) 6 (3) 2 (1) 15 (2)  115 (3)
5000 (-) 14 (1) 4 (2) 2 (2) 16 (2) 108 (15)
7500 (-) 16 (4) 5 (3) 3 (3) 13 (1) 110 (16)
10000 (-) 23 * (12) 3 (2) 2 (2) 14 (6) 106 (24)
               
167 (+) 15 (3) 5 (1) 15 (1) 32 (9) 138 (7)
500 (+) 34 *(13) 6 (3) 8 (4) 24 (13) 154 (16)
1670 (+) 63 *(32) 4 (1) 9 (4) 26 (10) 183 (31)
5000 (+) 109 *(20) 6 (5) 11 (3) 17 (10) 182 (4)
7500 (+) 116 * (43) 4 (1) 10 (4) 21 (8) 178 (37)
10000 (+) 66 * (26) 7 (2) 11 (8) 23 (6) 168 (19)

Data reported as: Mean (Standard Deviation).

*Positive response (≥ 2X solvent control value. Only two-fold or greater increases are indicated)

Apparently normal growth all strains/doses +/-S9.

No precipitate.

Applicant's summary and conclusion

Conclusions:
The test substance was found positive in the Ames/Salmonella Plate Incorporation Assay under the conditions, and according to the criteria, of the test protocol.

Executive summary:

The test substance Me-NENA was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay following a method similar to OECD Guideline 471 in a GLP study. The assay (plate incorporation method) was carried out in triplicate using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA1538 in the presence and absence of metabolic activation (S9 mixture prepared with 6% (v/v) Aroclor 1254-induced male sprague-Dawley rat liver homogenate). Dimethyl sulfoxide (DMSO) was used as solvent. Toxicity of the substance was first evaluated in a prescreen by treating duplicate cultures of strains TA1538 and TA100 at doses of 0.0 (solvent control), 50.0, 167, 500, 1670 and 5000 μg /plate in the absence of S9. The substance was found no toxic to each strain at all doses tested. In addition, the test substance was freely soluble at all doses evaluated. Based on these findings, the test substance was evaluated in the mutation assay at doses of 0.0 (solvent control), 167, 500, 1670, 5000, 7500 and 10000 µg/plate with and without S9. Triplicate cultures of each strain were evaluated with the solvent (negative control) and the appropriate positive control (sodium azide, 9-aminoacridine and 2-nitrofluorene for assay “without metabolic activation” and 2-anthramine for assay “with metabolic activation”) in the same conditions as that used for the test substance. A positive result was defined as a statistically significant, dose-dependent increase in the number of revertants with at least one dose level inducing a revertant frequency that is two-fold the spontaneous solvent control value. According to the results, statistically significant, dose-dependent increases in revertant frequencies were observed in strain TA1535 with (2.6-fold) and without S9 (1.6-fold). Thus, a confirmatory assay was conducted in the same conditions with the following results: statistically significant, dose-dependent increases in revertant frequencies were observed in strains TA1535 (13-fold) and TA100 (1.4-fold) with S9, and in strain TA1535 without S9 (2.0-fold). The mean values of revertant colonies of the negative (vehicle) control plates were within the historical control range. Also, positive control values were within acceptable limits. Based on the results of the study, it is concluded that the test item was positive in the Ames/Salmonella Plate Incorporation Assay under the conditions, and according to the criteria, of the test protocol.