Nonane-1,9-diol

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:

2003-09-03 to 2003-11-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Nonane-1,9-diol

EC Number:

223-517-5

EC Name:

Nonane-1,9-diol

Cas Number:

3937-56-2

Molecular formula:

C9H20O2

IUPAC Name:

nonane-1,9-diol

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Chemical name: 1,9-Nonanediol
- CAS no.: 3937-56-2
- Source and lot/batch No.of test material: : BASF / 42638
- Expiration date of the lot/batch: March 2003
- Molecular weight: 160.254 g/mol
- Purity: 98.8%

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced S9 (prepared in house)

Test concentrations with justification for top dose:

1st Experiment (plate incorporation) and Experiment 2 (pre-incubation): 0, 20, 100, 500, 2500, 5000 ug/plate (the maximum recommended concentration in accordance with current regulatory guidelines for in vitro bacterial genotoxicity assays)
Due to toxicity observed at 5000 ug/plate in Experiment 2, a further pre-incubation test was undertaken (Experiment 3): 0, 250, 500, 750, 1000 and 1500 ug/plate (maximum concentration limited by toxicity)

Vehicle / solvent:

Dimethyl sulphoxide (DMSO)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: MNNG, NOPD, AAC, 4-NQO

Details on test system and experimental conditions:

Test tubes containing 2 mL of soft agar (overlay agar) kept at 45°C in a water bath had the following added: 0.1 mL test article formulation (or vehicle) ; 0.1 mL fresh bacterial culture; 0.5 mL S9 mix (or PBS in the absence of metabolic activation).
After mixing Salmonella typhimurium containing tubes were poured onto Vogel-Bonner agar plates (minimal glucose agar plates). E.coli containing tubes were poured onto minimal agar plates.
Agar plates were incubated at 37°C for 48-72 h in the dark for the bacterial colonies (his+ or typ+ revertants) counted.
Pre-incubation:
0.1 mL of test article formulation (or vehicle), 0.1 mL bacterial suspension and 0.5 mL S9 mix (or PBS in the absence of metabolic activation) were incubated at 37°C for the duration of ~20 minutes. Subsequently, 2 mL of soft agar was added and samples were poured onto the agar plates.
In both cases, agar plates were incubated at 37°C for 48-72 h in the dark for the bacterial colonies (his+ or typ+ revertants) counted.

The background lawns of the plates were examined for signs of toxicity. Other toxicity indicators that may have been noted included a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response.

Evaluation criteria:

The test chemical was considered positive in this assay if the following criteria were met:
- dose-related and reproducible increase in the number of revertant colonies (i.e. doubling of the spontaneous mutation rate in at least one tester strain either –S9 or +S9)

A test substance was generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Statistics:

Statistics not warranted

Results and discussion

Test results

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

A. Mutation assay:

Experiment 1 (plate incorporation) treatments of all the tester strains were performed in the absence and presence of S-9, using final concentrations of 1,9-Nonanediol at 20, 100, 500, 2500, 5000 µg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity (evidenced by reduction in revertant numbers) was observed on the mutation plates treated at 5000 µg/mL with TA1537 and TA98 in the presence of S9. 1,9-Nonandiol was not mutagenic following a plate incorporation methodology

Experiment 2 (pre-incubation) treatments of all the tester strains were performed in the absence and presence of S-9 using the same dose concentrations as Experiment 1. Toxicity was evident at concentrations of 2500 ug/plate and greater. Consequently with only 4 scorable dose levels, a further pre-incubation experiment was undertaken to better define mutagenicity in the absence of overt toxicity.

Experiment 3 (pre-incubation) treatments of all the tester strains were performed in the absence and presence of S-9, using final concentrations of 1,9-Nonanediol at 250, 500, 750, 1000, 1500 µg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity (evidenced by both a reduction in revertant numbers and background lawn) was observed on the mutation plates treated at 1500 µg/mL with TA98 and WP2uvrA in the absence of S9. 1,9-Nonanediol was not mutagenic following a pre-incubation methodology

The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.

The positive controls induced an acceptable increase in revertant colony numbers,thereby demonstrating the sensitivity and specificity of the test system.

Following1,9-Nonandioltreatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were equal to or greater than 2-fold above the concurrent vehicle control. This study was therefore considered to have provided no evidence of any mutagenic activity in this assay system (refer to Table 7.6.1/01-1, -2, -3).

Table 7.6.1/01-1:
Bacterial (reverse) gene mutation plate incorporation data – Experiment 1

Conc (µg/plate)	TA98		TA100		TA1535			TA1537		WP2uvrA
	-S9	+S9	-S9	+S9	-S9	+S9		-S9	+S9	-S9	+S9
0	25	39	118	109	18	17		9	11	32	36
20	26	36	121	122	15	15		8	7	30	30
100	26	35	132	141	15	14		10	8	^29	^32
500	28	33	134	123	14	14		8	9	26	29
2500	23	36	127	128	10	10		6	6	24	29
5000	20	19	93	84	11	11		6	6	19	29
+ve	689	545	1117	986	907	128		459	113	614	253
+ve controls: -S9 (absence of metabolic activation): TA98: 4-nitro-o-phenylendiamine (NOPD) TA100, TA1535: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) TA1537: 9-aminoacridine (AAC) WP2uvrA: 4-nitroquinoline-N-oxide (4-NQO)							+S9 (presence of metabolic activation): All strains: 2-aminoanthracene

Table 7.6.1/01-2:
Bacterial (reverse) gene mutation pre-incubation data – Experiment 2

Conc (µg/plate)	TA98		TA100		TA1535			TA1537		WP2uvrA
	-S9	+S9	-S9	+S9	-S9	+S9		-S9	+S9	-S9	+S9
0	27	31	107	107	17	16		10	9	30	30
20	22	32	106	102	19	16		10	7	31	28
100	21	29	108	98	15	13		8	7	34	25
500	22	24	100	99	15	13		5	6	30	28
2500	10B	16B	43B	93B	2B	11B		1B	6B	26B	22
5000	0B	12B	0B	56B	0B	5B		0B	2B	0B	18B
+ve	802	603	805	846	719	91		379	118	634	242
B: reduced background lawn +ve controls: -S9 (absence of metabolic activation): TA98: 4-nitro-o-phenylendiamine (NOPD) TA100, TA1535: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) TA1537: 9-aminoacridine (AAC) WP2uvrA: 4-nitroquinoline-N-oxide (4-NQO)							+S9 (presence of metabolic activation): All strains: 2-aminoanthracene

Table 7.6.1/01-3:
Bacterial (reverse) gene mutation pre-incubation data – Experiment 3

Conc (µg/plate)	TA98		TA100		TA1535			TA1537		WP2uvrA
	-S9	+S9	-S9	+S9	-S9	+S9		-S9	+S9	-S9	+S9
0	26	32	108	116	19	16		8	9	30	37
250	22	27	102	110	18	15		8	8	26	37
500	30	34	112	104	16	13		6	7	20	30
750	21	31	102	102	14	14		7	7	26	29
1000	26	25	101	105	16	14		7	7	23	27
1500	14	19	101	75	14	10		5	5	12	22
+ve	620	792	717	842	717	106		415	126	628	232
+ve controls: -S9 (absence of metabolic activation): TA98: 4-nitro-o-phenylendiamine (NOPD) TA100, TA1535: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) TA1537: 9-aminoacridine (AAC) WP2uvrA: 4-nitroquinoline-N-oxide (4-NQO)							+S9 (presence of metabolic activation): All strains: 2-aminoanthracene

B. Deficiencies:

A single positive control, 2-aminoanthracene was used to evaluate the sensitivity of the assay in both the presence of S9-mix. This positive control may be activated by enzymes other than the microsomal cytochrome P450 family. Therefore it could be concluded that the S9 activity has not been demonstrated. However, S9 was prepared in house and the efficacy of the S9 mix was characterised with benzo[a]pyrene. Overall it is concluded that the S9 activity was adequately demonstrated.

Applicant's summary and conclusion

Conclusions:

It was concluded that 1,9-Nonanediol did not induce mutation in four histidine-requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium, and one tryptophan-requiring strain (WP2uvrA) of Escherichia coli when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/mL (the maximum recommended concentration according to current regulatory guidelines, and in several cases a toxic treatment concentration), in the absence and presence of a rat liver metabolic activation system (S9) using both plate incorporation and pre-incubation methodologies.

Executive summary:

In a reverse gene mutation assay in bacteria, S. typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2uvrA were exposed to1,9-Nonanediol formulated in dimethyl sulphoxide.Both plate incorporation and pre-incubation methodologies were used.

Experiment 1 (plate incorporation) treatments of all the tester strains were performed in the absence and presence of S-9, using final concentrations of 1,9-Nonandiol at 20, 100, 500, 2500, 5000 µg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity (evidenced by reduction in revertant numbers) was observed on the mutation plates treated at 5000 µg/mL with TA1537 and TA98 in the presence of S9. 1,9-Nonandiol was not mutagenic following a plate incorporation methodology

Experiment 2 (pre-incubation) treatments of all the tester strains were performed in the absence and presence of S-9 using the same dose concentrations as Experiment 1. Toxicity was evident at concentrations of 2500 ug/plate and greater. Consequently with only 4 scorable dose levels, a further pre-incubation experiment was undertaken to better define mutagenicity in the absence of overt toxicity.

Experiment 3 (pre-incubation) treatments of all the tester strains were performed in the absence and presence of S-9, using final concentrations of 1,9-Nonandiol at 250, 500, 750, 1000, 1500 µg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity (evidenced by both a reduction in revertant numbers and background lawn) was observed on the mutation plates treated at 1500 µg/mL with TA98 and WP2uvrA in the absence of S9. 1,9-Nonandiol was not mutagenic following a pre-incubation methodology

The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.

The positive controls induced an acceptable increase in revertant colony numbers, thereby demonstrating the sensitivity and specificity of the test system.

Following 1,9-Nonanediol treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were equal to or greater than 2-fold above the concurrent vehicle control. This study was therefore considered to have provided no evidence of any mutagenic activity in this assay system.

It was concluded that 1,9-Nonanediol did not induce mutation in four histidine-requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium, and one tryptophan-requiring strain (WP2uvrA) of Escherichia coli when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/mL (the maximum recommended concentration according to current regulatory guidelines, and in several cases a toxic treatment concentration), in the absence and presence of a rat liver metabolic activation system (S9) using both plate incorporation and pre-incubation methodologies.