Nonanal

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:

02 December 1980 to 08 December 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Meets generally accepted scientific standards with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1980

Report date:

1981

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

The bacterial strains tested differ slightly from the guideline.

GLP compliance:

no

Remarks:

Study predates GLP

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Nonanal

EC Number:

204-688-5

EC Name:

Nonanal

Cas Number:

124-19-6

Molecular formula:

C9H18O

IUPAC Name:

nonanal

Specific details on test material used for the study:

- Name of test material (as cited in study report): C192 SN 2188-2; Aldehyde C9 Nonylic
- Appearance: Clear colourless liquid

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal enzyme preparations from Aroclor induced rats

Test concentrations with justification for top dose:

Initial assay: 0, 0.01, 0.1, 1.0, 5.0, 10.0, 25.0 and 50.0 µL per plate
Definitive assay: 0, 0.0001, 0.0005, 0.001, 0.005 and 0.01 µL per plate

Vehicle / solvent:

- Solvent used: DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA-1537: without metabolic activation; 50 µg/plate

Controls 2

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA-1535 and TA-100: without metabolic activation; 10 µg/plate

Controls 3

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA-1538 and TA-98: without metabolic activation; 10 µg/plate

Controls 4

Positive controls:

yes

Positive control substance:

other: 2-anthramine, all strains: with metabolic activation; 2.5 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

MEDIA
The bacterial strains were cultured in Oxoid Media #2 (nutrient broth). The selective medium was Vogel Bonner Medium E with 2% glucose. The overlay agar consisted of 0.6% purified agar with 0.5 mM histidine, 0.05 mM biotin and 0.1 M NaCl.

METABOLIC ACTIVATION SYSTEM
Preparation of S9 homogenate:
- A 9000xg supernatant prepared from Sprague-Dawley adult male rat liver induced by Aroclor 1254 was used in this assay.
Preparation of S9-Mix:
- S9-mix contained, per mL, 4 µmoles NADP, 5 µmoles D-glucose-6-phosphate, 100 µmoles sodium phosphate buffer pH 7.4; 8 µmoles MgCl₂, 33 µmoles KCl and 100 µL organ homogenate from rat liver (S9 fraction).

TEST PROCEDURE
a). Non-activation assay:
The following was added to a sterile 13x100 mm test tube placed in a 43 °C water bath:
- 2 mL of 0.6% agar containing 0.05 mM histidine and 0.05 mM biotin
- 0.05 mL of a solution of the test chemical to give the appropriate dose
- 0.1 - 0.2 mL of the indicator organism
- 0.5 mL of 0.2 M phosphate buffer, pH 7.4
The mixture was swirled gently and then poured onto minimal agar plates. After the top agar had set, the plates were incubated at 37 °C for approximately 2 days. The number of his+ revertant colonies growing on the plates was counted and recorded.
b). Activation assay:
The activation assay was run concurrently with the non-activation assay. The only difference was the addition of 0.5 mL of S9 mix to the tubes in the place of 0.5 mL of phosphate buffer.

Evaluation criteria:

1). Strains TA-1535, TA-1537 and TA-1538:
If the solvent control value was within the normal range, a test material producing a positive response equal to 3 times the solvent control is considered mutagenic.

2). Strains TA-98 and TA-100:
If the solvent control value was within the normal range, a test material producing a positive response equal to twice the solvent control is considered mutagenic.

If a test material produced a response in a single test which couldn't be reproduced in additional runs, the initial positive test data loses significance.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test material was toxic to the strains TA-1537 and TA-1538 at doses of 0.01 µL and above and to TA-98 at doses of 1.0 µL and above.

In the initial assays, indicator strains TA-1535 and TA-100 were not used because these 2 strains were contaminated with other types of bacteria. The test was subsequently repeated with all the indicator strains in the nonactivation and activation assays and the dose range employed was from 0.0001 µL to 0.01 µL per plate based on the toxicity observed with the strains TA-537, TA-1538 and TA-98. The test material was toxic to all strains except TA-98 and TA-100 at 0.01 µL dose in the repeat test.

The results of the tests conducted on the compound in both the presence and absence of a metabolic system were negative.

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

Table 1: Initial assay

Test	Concentration (µL per plate)	Revertants per plate
		TA-1537	TA-1538	TA-98
Non-activation
Solvent control	-	5	14	25
Solvent control	-	7	16	31
Positive control *	-	229	1261	828
Positive control *	-	234	1336	924
Test compound	0.01	0	0	27
	0.1	0	0	28
	1.0	1	0	0
	5.0	0	0	0
	10.0	0	0	0
	25.0	0	0	0
	50.0	0	0	0
Activation
Solvent control	-	9	25	32
Solvent control	-	9	30	51
Positive control **	-	141	1028	1143
Positive control **	-	203	1625	1193
Test compound	0.01	3	18	24
	0.1	5	14	27
	1.0	1	0	2
	5.0	0	0	0
	10.0	0	0	0
	25.0	0	0	0
	50.0	0	0	0

*:

TA-1537: 9-aminoacridine (50 µg/plate)

TA-1538: 2-nitrofluorene (10 µg/plate)

TA-98: 2-nitrofluorene (10 µg/plate)

 

**:

TA-1537: 2-anthramine (2.5 µg/plate)

TA-1538: 2-anthramine (2.5 µg/plate)

TA-98: 2-anthramine (2.5 µg/plate)

 

Solvent: 50 µL/plate

 

 

Table 2: Definitive assay

Test	Concentration (µL per plate)	Revertants per plate
		TA-1535	TA-1537	TA-1538	TA-98	TA-100
Non-activation
Solvent control	-	27	10	18	34	117
Solvent control	-	29	14	10	46	120
Positive control *	-	1468	463	1445	704	950
Positive control *	-	1497	520	1396	717	1075
Test compound	0.0001	24	13	12	29	113
	0.0005	22	9	13	22	123
	0.001	27	11	16	27	124
	0.005	21	9	18	31	120
	0.01	0	0	0	21	86
Activation
Solvent control	-	24	11	13	32	112
Solvent control	-	26	12	25	34	115
Positive control **	-	233	295	1681	629	1852
Positive control **	-	204	285	1135	1216	1711
Test compound	0.0001	29	12	25	30	116
	0.0005	24	13	24	36	124
	0.001	24	11	18	27	123
	0.005	29	12	20	20	128
	0.01	12	5	11	21	102

*:

TA-1535: sodium azide (10 µg/plate)

TA-1537: 9-aminoacridine (50 µg/plate)

TA-1538: 2-nitrofluorene (10 µg/plate)

TA-98: 2-nitrofluorene (10 µg/plate)

TA-100: sodium azide (10 µg/plate)

 

**:

TA-1535: 2-anthramine (2.5 µg/plate)

TA-1537: 2-anthramine (2.5 µg/plate)

TA-1538: 2-anthramine (2.5 µg/plate)

TA-98: 2-anthramine (2.5 µg/plate)

TA-100: 2-anthramine (2.5 µg/plate)

 

Solvent: 50 µL/plate

 

Applicant's summary and conclusion

Conclusions:

The test substance was assessed for genotoxicity according to OECD 471 with and without metabolic activation. The test material did not exhibit genetic activity in any of the assays conducted in this evaluation and was considered not mutagenic under the test conditions.