2-nitrobenzaldehyde

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

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

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.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

equivalent or similar to guideline

Guideline:

other: Rec assay

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

Species / strain / cell type:

S. typhimurium TA 1538

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

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.

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

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'.

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Under the condition of the test, the TS has not to be classified according to CLP.

Four Ames Tests with the substance o-Nitrobenzaldehyde from the literature are available. Three (Shimizu 1986, Suzuki 1983, Kawai 1987) of them show negative results and one (Chiu 1978) a positive result.

The study published by Shimizu (1986) was the most reliable study and was selected as key.

Shimizu (1986) analyzed five bacterial strains of Salmonella thyphimurium (TA 98, TA 1538, TA 1537, TA 100 and TA 135) to test the substance in a concentration range from 0.01 mg/plate to 5 mg/plate using six concentrations. Cytotoxic effects were observed above 0.5 mg substance per plate. In addition, a rec-assay with Bacillus subtilis (H17 and M45) was conducted simultaneously. The combination of both test systems verified very efficiently the absence of DNA damaging effects of the test substances. The method of this publication was described very well and was similar to the requirements of the OECD test guideline 471. In conclusion, the negative test result was considered to be reliable.

The result described by Suzuki (1983) supports this negative result. No mutagenic effects occurred to the two test strains TA 98 and TA 100 up to the highest tested concentration of 100 µg/plate under conditions similar to OECD 471. However, the analysis of only two strains and the lack of positive control data restrict the reliability of this study.

The study by Kawai (1987) also supports the negative result using the two test strains TA 98 and TA 100. Beside the result, no further data were extractable from the article as the major part of this literature is published in original language (Japanese) and thus the reliability of this source is considered as not assignable.

In contrast, Chiu (1978) analyzed two Salmonella thyphimurium strains (TA 98, TA 100) in a concentration range from 0.1 µmole (0.015 mg) to 1 µmole (0.15 mg) o-Nitrobenzaldehyde per plate and found positive AMES test results only for the test strain TA 98 without metabolic activation at concentrations of 1 µmole (0.15 mg) and 10 µmole (1.51 mg) per plate. Contrary to the OECD guideline 471, no tests with further bacterial test strains or with metabolic activation were conducted. Because these effect concentrations for mutagenicity are in the same range as the observed cytotoxicity effect concentrations published in the key study by Shimizu (1986) and because no purity of the test substance was provided, this study was considered as not reliable.

Based on the more reliable and supported negative mutagenic results it is concluded that the substance does not induce mutation in bacterial cells.

 

 

Justification for selection of genetic toxicity endpoint
Four Ames Tests with the substance o-Nitrobenzaldehyde from the literature are available. Three (Shimizu 1986, Suzuki 1983, Kawai 1987) of them show negative results and one (Chiu 1978) a positive result.
The study published by Shimizu (1986) was the most reliable study and was selected as key.
The study of Chiu (1978) presenting the positive mutagenic effects was considered as not reliable because the test was conducted contrary to the OECD guideline 471 testing only two bacterial test strains without metabolic activation. Furthermore, the observed effect concentrations for mutagenicity are in the same range as the observed cytotoxicity effect concentrations published in the key study by Shimizu (1986) and no purity of the test substance was provided in this study.
Based on the more reliable and supported negative mutagenic results it is concluded that the substance does not induce mutation in bacterial cells.