2-nitrobenzaldehyde

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Data from the literature in which all parameters described are closely comparable to a guideline method and well documented.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1986

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

other: Rec assay

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

o-Nitrobenzaldehyde

IUPAC Name:

o-Nitrobenzaldehyde

Constituent 2

Reference substance name:

2-nitrobenzaldehyde

EC Number:

209-025-3

EC Name:

2-nitrobenzaldehyde

Cas Number:

552-89-6

Molecular formula:

C7H5NO3

IUPAC Name:

2-nitrobenzaldehyde

Details on test material:

- Name of test material (as cited in study report): o-Nitrobenzaldehyde
- Analytical purity: min. 99%
- Source: Tokyo Kasei Kogyo Co.

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain 3

Species / strain / cell type:

bacteria, other: Bacillus subtilis

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0.01, 0.05, 0.1, 0.5, 1, 5 mg (Dose/plate)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterilized dimethyl sulphoxide (DMSO)
- Source: Junsei Chemical Co

Controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

other: N-Ethyl-N'- nitro-N-nitrosoguanidine (ENNG), 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

Ames test:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period: 0.1 ml of various concentrations of test compounds were added to a sterile test-tube containing 3 -6 x 10^7 bacerial cells, 0.5 ml of S9 mix or sodium phosphate buffer (pH 7.4). This mixture was preincubated in a shaker water-bath at 37°C for 15 min, then added to 2 ml molten top agar (45°C). The contents of each tube were mixed well and immediately poured onto the surface of a minimal-agar plate.
- Exposure duration: The plates were inverted and incubated at 37°C in the dark for 70 h.
- Control: A control plate contained 0.05 ml of DMSO.
- Contamination test: was carried out through each experiment.
- The background bacterial lawn: was routinely checked by microscopy
- Ampicilin resistance: TA 98 and TA 100 were checked routinely

NUMBER OF REPLICATIONS: all tests were performed in duplicate and repeated at least 3 times separately

Rec assay:

Rec assay according to procedures described by Kada et al. (1972) and Hirano et al. (1982).
In brief, liquid broth medium consisted of 10 g beef extract, 10 g polypeptone and 5 g NaCl per 100 ml. Minimal salt solution (MM) containing 1 g (NH4)2SO4, 10 g KH2PO4, 0.1 g MgSO4 x 7H2O and 0.5 g sodium citrate per 1000 ml (neutralized with KOH) was used for dilution and washing of bacteria. Overnight cultures of strains H17 and M45 in liquid broth medium were diluted 10 times with minimal salt solution. They were streaked from small pipettes onto the surface of a broth agar plate, care being taken not to let them touch one another. Paper discs, 13 mm in diameter, were prepared by punching filter papers. A disc has soaked in 0.05 ml of various concentrations of compounds and placed on the plate so as to cover the end of the bacterial streaks. After incubation for 24 h at 37°C , grown bacteria become visible except in the inhibition zone depending on the strain and on the compound used. When a compound showed higher inhibition to M45 (Rec-) than to H17 (Rec+) the assay was repeated. A difference of more than 1 mm was a positive response.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA 98, TA 1538, TA 1537, TA 100, TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Concentrations 1 and 5 mg/plate indicate toxic effects with and without metabolic activation in all test strains

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

other: Bacillus subtilis

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 2: Results for o-Nitrobenzaldehyde, data represent the results of 3 separate experiments and give the mean values of 3 plates. The numbers of revertant colonies indicate mean +- S.D., all these numbers of revertant colonies were counted after 68 - 72 h incubation.

Dose (/plate)	Ames test										Rec assay
	TA98		TA1538		TA1537		TA100		TA1535		(mm)**
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0.01 mg	24 +-3	22 +-3	20 +-3	21 +-3	8 +-2	10 +-2	163 +-18	174 +-20	31 +-5	35 +-4
0.05	28 +-4	31 +-5	28 +-3	26 +-4	9 +-2	9 +-1	168 +-22	170 +-23	21 +-3	30 +-5
0.1	24 +-3	28 +-4	31 +-3	25 +-4	12 +-3	11 +-2	164 +-16	168 +-19	29 +-5	32 +-4
0.5	14 +-7*	38 +-5	18 +-2	22 +-3	10 +-3	10 +-2	169 +-18	173 +-16	35 +-5	32 +-4	0
1	0*	10 +-4*	0 *	0*	0*	0*	0*	0*	0*	0*
5	0*	0*	0*	0*	0*	0*	0*	0*	0*	0*	5.5
DMSO
0.05 ml	26 +-5	28 +-6	22 +-4	20 +-5	8 +-3	10 +-2	181 +-26	191 +-29	30 +-5	34 +-4	0
ENNG
2 µg							1778 +-401
10 µg									1389 +-331
2 -NF
2 µg	1559 +-325
5 µg			1632 +-294
9 -AA
100 µg					1068 +-447
2 -AA		1014 +-166						1322 +-238
2 µg
5 µg				1082 +-351		185 +-41				161 +-29
4 -NQO
1 mg											7

*Indicates toxic effects; **Indicates the length of inhibition zones for rec (M45)-rec (H17) strains of B. subtilis; 0 Indicates no revertants detectable; Control: average value of all control groups in each assay

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under this test conditions no mutagenic effects of o-Nitrobenzaldehyde were observed.

Executive summary:

The mutagenicities of 37 mono-nitrobenzene derivatives, i.e. the ortho, meta, and para isomers of nitrotoluene, nitrophenol, nitroaniline, nitroanisole, nitrobenzaldehyde, nitrobenzyl chloride, nitrobenzonitrile, nitroacetophenone, nitrophenetol, nitrobenzoic acid, nitrophenylacetic acid, and nitrocinnamic acid and p-nitrothiophenol, were tested in the Ames test and rec assay. The rec assay gave more positive results than the Ames test. The para-substituted nitrobenzene derivatives were always positive in the rec assay as were all but 2 in the Ames test, while 5 out of 12 ortho and meta derivatives were mutagenic in the Ames test. The results of the present study, combined with those of the previous study, are discussed with special reference to the structure-mutagenicity relationships.

In this endpoint study record only the results for the substance o-Nitrobenzaldehyde (CAS 552 -89 -6) were considered.