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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Nitromethane was negative in all in vitro and in vivo tests conducted except for the SHE cell transformation assay.  The transformation assay is known to respond to many different mechanisms including 'nonmutagenic' mechanisms that might not be expected to induce micronuclei.  No mutagenic activity was observed with or without mutagenic activity in the in vitro mammalian cell transformation assay using an appropriate surrogate, nitroethane.  See IUCLID Chapter 13 for read across justification.

Link to relevant study records
Reference
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:
circa 1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to guideline and/or standard method but was non-GLP.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only used Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S-9 was prepared from liver homogenate from male Sprague-Dawley rats and male Syrian hamsters that had been injected with Aroclor 1254 (500 mg/kg) 5 days before they were killed.
Test concentrations with justification for top dose:
100, 333.3, 1000, 3333.3 and 10000 micrograms/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: With: 2-aminoanthracene was tested on all strains in the presence of rat and hamster S-9. Without: 4-nitro-o-phenylenediamine was tested on TA98, sodium azide was tested on TA100 and TA1535, and 9-aminoacridine was tested on TA1537.
Details on test system and experimental conditions:
Bacteria: All strains of bacteria were supplied by the same supplier (Dr. B. Ames). Cultures were kept frozen at -70 degrees C until use. The day before the test, the entire 1-ml sample was thawed and inoculated into minimal glucose medium. The culture was grown overnight for 12-15 hours at 37 degrees C on a shaker, and the phenotype was analyzed.

Test material: The test material was coded and tested as an unknown. Test material was dissolved in DMSO. A preliminary study with 10000 micrograms/plate was conducted in strain TA100 to determine if this concentration was toxic. Since it did not cause toxicity or precipitate, this concentration was chosen as the upper limit for the study. Six concentrations of test compound (100, 333, 1000, 3333, 6666 and 10000 micrograms/plate) were used in the test.

The positive control 2-aminoanthracene was tested on all strains in the presence of rat and hamster S-9. 4-nitro-o-phenylenediamine was tested on TA98, sodium azide was tested on TA100 and TA1535, and 9-aminoacridine was tested on TA1537 without S-9. The concentrations used were not listed, but were referenced (Haworth et al., Environ Mutagen 5(Suppl 1):3-142, 1983).

S-9 mix: S-9 was prepared from liver homogenate from male Sprague-Dawley rats and male Syrian hamsters that had been injected with Aroclor 1254 (500 mg/kg) 5 days before they were killed. Food was withheld from the animals 12-24 hours prior to euthanization. The S-9 fraction was obtained by centrifuging the liver homogenate for 10 min (4 degrees C) at 9000 g. It was stored at -70 degrees C until use. The S-9 mix was prepared immediately before use. It contained 0.10 ml S-9 fraction, 0.02 ml 0.04 M MgCl2, 0.02 ml 1.65 M KCl, 0.10 ml 0.04 M NADP, 0.10 ml 0.05 M glucose-6- phosphate, 0.10 ml 1.0 M NaH2PO4 (pH 7.4) and 0.56 ml distilled water. The concentration of S-9 used in experiments was 10%.

Study conduct: All tests were performed in triplicate. To each of 13 x 100 mm test tubes, the following chemicals were added: 0.5 ml of S-9 mix (for metabolic activation) or 0.1 M PO4 buffer (pH 7.4), 0.05 ml of the overnight culture, and 0.05 ml of DMSO (negative control), test material or positive control. The mixture was incubated at 37 degrees for 20 minutes (without shaking), and 2.0 ml of molten (45 degrees C) top agar supplemented with 0.5 mM L-histidine and 0.5 mM D-biotin was added. The contents of each tube were mixed and immediately poured onto the surface of a Vogel-Bonner plate. Plates were inverted and incubated at 37 degrees C for 48 hr. Colonies of his+ revertants were then counted as described by Haworth et al. (Environ Mutagen 5(Suppl 1):3-142, 1983). The test was repeated no less than one week later.

Each shipment of chemicals to the testing laboratories was accompanied by information on the volatility, density, solubility, flammability, stability, and storage conditions for each chemical, as well as by safety instructions. It's unclear whether the testing laboratories conducted the test on nitromethane in a closed system.

Evaluation criteria:
The criteria used for evaluation were the same as those described by Haworth et al. A mutagenic response was characterized by a dose-related increase in the number of revertants with respect to the negative control (even if the increase was less than twofold. The material was not mutagenic if there was no increase in the number of mutants. The response was questionable if there was not a clear dose response relationship, if a dose-related relationship was not reproducible, or if the response was of insufficient magnitude to support a positive response.
Statistics:
No additional information available.
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:
cytotoxicity
Remarks:
Observed in strain TA100 at 10,000 ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
There was no effect of test material on the incidence of mutations. No dose-response effect was observed. Toxicity was observed in strain TA100 at 10,000 ug/plate. . Metabolic activation with rat or hamster S-9 did not appear to significantly increase the number of mutations observed at each concentration.

The average number of revertants in the controls for strains TA98, TA100, TA1535 and TA1537 in the absence of S-9 were 28, 82, 23 and 8. The number of revertants observed in cultures treated with test material in the absence of S9 ranged from 25-37 in TA98, 92-127 in TA100, 19-23 in TA1535 and 7-9 in TA1537.

In the presence of 10 % hamster S-9, the average number of revertants in the controls for strains TA98, TA100, TA1535 and TA1537 were 40, 104, 11 and 11, respectively. In the presence of 10% hamster S-9, the number of revertants observed in cultures treated with test material ranged from 33-44 in strain TA98, 101-120 in TA100, 10-14 in TA1535, and 12-15 in TA1537.

In the presence of 10 % rat S-9, the average number of revertants in the controls for strains TA98, TA100, TA1535 and TA1537 were 48, 101, 9 and 12, respectively. In the presence of 10% rat S-9, the number of revertants observed in cultures treated with test material ranged from 37-48 in TA98, 89-109 in TA100, 9-14 in TA1535, and 2-5 in TA1537.

Incidences in the positive control cultures ranged from 221 (in TA1537 with 10% rat liver S-9) to 1720 (in TA98 with 10% hamster liver S-9). The numbers of revertants in the positive controls were at least 5 times greater than the negative controls.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Mutagenicity of Nitromethane in Salmonella typhimurium

                    Revertants/plate
        -S9     +10% hamster S9     +10% rat S9
 Strain  Dose(ug/plate)  Trial 1  Trial 2  Trial 1  Trial 2  Trial 1  Trial 2
 TA100  0  64 + 1.9  82 + 2.8  54 + 1.7  104 + 6.8  55 + 2.3  101 + 6.1
   100  70 + 2.3  104 + 2.2  92 + 11.4  113 + 7.5  86 + 5.5  109 + 11.0
   333.3  74 + 3.2  106 + 10.3  93 + 4.4  111 + 0.6  88 + 2.1  89 + 4.7
   1,000  64 + 1.8  92 + 4.5  85 + 4.3  101 + 8.7  88 + 0.6  94 + 5.5
   3,333.3  60 + 4.2  101 + 11.3  90 + 6.7  105 + 10.0  91 + 10.3  101 + 8.4
   10,000  27 + 15.3*  127 + 9.1  96 + 5.5  120 + 3.2  84 + 5.7  99 + 6.1
 Positive Control    222 + 20.9  461 + 5.9  2168 + 100.8  1720 + 67.7  738 + 28.9  577 + 26.1 
 TA1535  0  7 + 1.2  23 + 2.0  6 + 2.3  11 + 1.5  8 + 0.6  9 + 1.2
   100  7 + 1.5  19 + 2.6  8 + 2.9  10 + 2.8  5 + 0.9  13 + 2.8
   333.3  9 + 1.2  19 + 1.3  5 + 1.0  10 + 1.5  6 + 1.5  13 + 2.1
   1,000  10 + 1.2  21 + 2.0  4 + 1.0  11 + 3.2  5 + 0.9  9 + 2.0
   3,333.3  12 + 2.7  20 + 3.0  4 + 2.0  12 + 1.8  6 + 0.6  10 + 1.9
   10,000  5 + 2.0  23 + 1.5  5 + 1.5  14 + 3.1  11 + 1.0  14 + 1.3
 Positive control    248 + 13.6  458 + 19.8  322 + 52.9  421 + 16.5  332 + 24.6  392 + 23.1
 TA1537  0  8 + 1.2  8 + 2.6  4 + 1.5  11 + 0.9  6 + 2.2  12 + 2.2
   100  3 + 1.3  7 + 0.9  5 + 0.9  13 + 2.6  6 + 0.7  4 + 1.5
   333.3  4 + 0.7  7 + 1.2  4 + 0.7  12 + 3.2  5 + 0.6  4 + 1.5
   1,000  3 + 1.5  8 + 1.0  2 + 0.0  13 + 2.6  6 + 1.0  5 + 0.3
   3,333.3  1 + 1.0  9 + 1.7  5 + 1.8  15 + 2.1  6 + 0.9  3 + 0.6
   10,000  1 + 0.7  7 + 3.0  6 + 1.2  12 + 1.9  3 + 0.7  2 + 0.6
 Positive control    530 + 132.9  431 + 20.9  152 + 22.0  510 + 10.7  298 + 20.9  221 + 31.0
 TA98  0  26 + 4.4  28 + 1.5  24 + 3.5  40 + 1.9  21 + 2.6  48 + 4.3
   100  19 + 0.3  37 + 0.3  28 + 1.5  43 + 6.2  33 + 4.2  48 + 3.6
   333.3  17 + 4.9  34 + 4.3  32 + 4.4  33 + 5.6  25 + 5.8  43 + 2.0
   1,000  16 + 2.6  31 + 2.8  27 + 1.8  44 + 1.3  30 + 4.7  47 + 4.5
   3,333.3  21 + 2.6  25 + 2.6  33 + 2.9  41 + 0.9  26 + 5.5  37 + 3.1
   10,000  16 + 1.5  30 + 5.2  26 + 2.7  36 + 5.7  26 + 0.9  39 + 1.2
 Positive control     837 + 116.2   777 + 23.2   1733 + 92.8   1598 + 76.2  458 + 13.6  511 + 35.6

* Slight toxicity

The positive controls in the absence of metabolic activation were sodium azide (TA100 and TA1535), 9-aminoacridine (TA1537), and 4-nitro-o-phenylenediamine (TA98). The positive control for metabolic activation with all strains was 2-aminoanthracene.

Conclusions:
Interpretation of results (migrated information):
negative

Nitromethane was negative in the Ames test with and without metabolic activation at concentrations as high as 10 mg/plate.
Executive summary:

Nitromethane was evaluated in the bacterial reverse mutation assay (Ames test). Nitromethane was negative in the Ames test with and without metabolic activation at concentrations as high as 10 mg/plate.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

In vitro studies

The mutagenic activity of nitromethane was evaluated in the Ames test using strains TA98, TA100, TA1535, TA1537 and TA1538. No mutagenic response was seen in the presence or absence of S-9 mix at 23,732 ppm (118.7 mg/L) nitromethane, approximately one half of a saturated atmosphere. Toxicity was noted in strains TA1535 and TA1537 at 47,465 ppm nitromethane. Nitromethane was negative in the Chinese Hamster Ovary Chromosomal Aberration Test with and without metabolic activation at doses as high as 4980 micrograms/ml. Nitromethane was negative in the SHE micronucleus assay. In the Syrian hamster embryo cell transformation assay, nitromethane produced positive results. For the in vitro mammalian gene mutation assay, nitroethane was used for read across for nitromethane (see IUCLID chapter 13 for read across justification). Nitroethane was evaluated in the in vitro Chinese hamster ovary cell/hypoxanthineguanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay. The genotoxic potential of the test material was assessed in two independent assays in the absence and presence of a metabolic activation (S9) system. The concentrations ranged from 0 (solvent control) to 751 μg/ml in the absence of S9 and in the presence of S9. The highest concentration was based on the guideline limit of 10 mM for this assay system. The results of this CHO/HGPRT forward gene mutation assay indicate that nitroethane was non-mutagenic when evaluated in the absence or presence of an externally supplied metabolic activation (S9) system.

 

In vivo studies

Nitromethane was negative in the mouse bone marrow micronucleus assay at ip doses as high as 1830 mg/kg or following 13 weeks exposure at concentrations as high as 1,500 ppm. In the Drosophila Basc test, nitromethane was negative at 125mM, a dose very close to the LD50.

Justification for selection of genetic toxicity endpoint

The study was conducted according to guideline and/or standard method but was non-GLP.  

Justification for classification or non-classification

In vitro and in vivo genotoxicity testing indicates a lack of mutagenic activity for nitromethane, therefore, no classifiication for genotoxicity is required.