cyclohexadecanone

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:

03 Mar - 06 Apr 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

Commission Directive 92/69/EEC

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

MINISTERIUM FÜR RAUMORDNUNG UND UMWELT DES LANDES SACHSEN-ANHALT, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

438-930-8

EC Name:

-

Cas Number:

2550-52-9

Molecular formula:

C16H30O

IUPAC Name:

cyclohexadecanone

Method

Target gene:

his operon (S. typhimurium strains)
trp operon (E. coli strain)

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:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital and ß-Naphthoflavone

Test concentrations with justification for top dose:

Range-finding study:
10, 50, 100, 500, 1000 and 5000 µg/plate with and without metabolic activation in TA 100 and WP2 uvr A

Experiment 1:
10, 50, 100, 500, 1000 and 5000 µg/plate with and without metabolic activation in TA 1537, TA 1535 and TA 98

Experiment 2:
10, 50, 100, 500, 1000 and 5000 µg/plate with and without metabolic activation in all strains

Top dose in experiment 1 and 2 was selected based on the precipitation of the test substance at 5000 µg/plate, which interfered with the scoring in the range-finding study.

Vehicle / solvent:

- Vehicle/Solvent used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: experiment 1: in agar (plate incorporation); experiment 2: preincubation

DURATION
- Preincubation period: 20 min (experiment 2)
- Exposure duration: 48 h (experiment 1 and 2)

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: determination of bacterial background lawn

Evaluation criteria:

A test is considered to be positive if the test item induces dose related increases in numbers of revertants scored in two separate experiments and these increases are deemed to be of biological relevance. Reproducible increases at one experimental point may also indicate a positive response. For a biologically relevant response the number of revertants is expected to be at least double the spontaneous reversion rate in the S. typhimurium strains TA 98, TA 100 and the E. coli strain. In the S. typhimurium strains TA 1535 and TA 1537 the number of revertants is expected to be at least the triple of the spontaneous reversion rate. A test item producing neither a dose related and reproducible increase in the number of revertants nor a reproducible positive response at any experimental point is considered to be non-mutagenic in this test system.

Statistics:

Mean values and standard deviations were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

(at 5000 µg/plate in range-finding study, reported as experiment 1)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

(at 5000 µg/plate in range-finding study, reported as experiment 1)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the toxicity range-finding study at a concentration of 5 mg/mL the test material precipitated in the cell culture medium.

RANGE-FINDING/SCREENING STUDIES: A toxicity range-finding study was carried out to determine the maximum concentration of the test substance in the main experiments. Therefore, S. typhimurium strain TA 100 and E. coli WP2 uvr A were tested at concentrations of 10, 50, 100, 500, 1000 and 5000 µg/plate with and without metabolic activation. A precipitation of the test substance, which interfered with the scoring, was observerd at 5000 µg/plate. Based on this result concentrations of 10, 50, 100, 500 and 1000 µg/plate were chosen for the main experiments. The results of the range-finding study for TA 100 and WP2 uvr A are used for experiment 1.

HISTORICAL CONTROL DATA
In both independent experiments controls gave counts of spontaneous revertants within the normal ranges obtained in this laboratory, except of one untreated control of WP2 uvr A. However, these value is in the normal range of spontaneous revertants of WP2 uvr A (30 - 60) according to literature*. The spontaneous revertants of the vehicle control was within the normal range. *(Green and Muriel (1976), Mutation Res. 38, 3-32)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Bacterial background lawn indicated sufficient growth conditions in all experiments.

Any other information on results incl. tables

Table 1: Test Results of Experiment 1

EXPERIMENT 1 (plate incubation method)
S9-Mix	Without
Test item (mg/plate)	TA 1535	TA 1537	TA 98	TA 100	WP2 uvr A
NC	16.0±5.3	11.3±3.2	22.0±3.0	239.0±31.4	39.0±4.2
SC	16.0±6.6	13.7±4.2	22.0±5.6	207.0±12.0	23.0±1.0
0.01	12.7±4.9	13.3±2.3	16.3±3.2	251.0±11.3	27.3±5.7
0.05	13.0±1.7	11.0±4.4	18.0±4.6	213.0±8.9	25.3±5.7
0.1	8.7±2.5	13.0±1.7	20.3±3.5	191.0±6.2	26.7±4.9
0.5	10.3±3.1	8.0±1.7	15.0±1.7	221.7±25.2	31.7±6.5
1	10.0±3.5	8.3±2.5	13.7±3.2	209.0±10.4	26.3±9.1
5				226.0±28.7	21.7±5.7
NaN3	1818.7±546.0			2490.7±187.5
9-AA		676.0±511.6
2-NF			585.3±102.1
MMS					118.0±23.3
S9-Mix	With
Test item (mg/plate)	TA 1535	TA 1537	TA 98	TA 100	WP2 uvr A
NC	16.3±5.5	13.0±7.0	22.3±5.7	255.0±18.7	36.7±2.5
SC	16.0±2.6	14.0±1.0	20.7±8.1	221.3±22.5	31.3±4.0
0.01	14.7±9.0	15.7±5.9	16.3±2.1	234.0±19.3	29.7±11.6
0.05	14.3±3.2	11.7±3.5	21.7±2.1	246.0±9.2	34.7±2.5
0.1	12.3±4.0	12.7±3.2	16.7±5.5	222.3±27.1	33.0±3.5
0.5	13.0±4.4	10.3±2.3	16.0±8.2	257.7±34.0	30.0±5.3
1	9.3±1.5	12.0±3.6	17.7±3.2	226.7±16.5	30.00±5.3
5				212.0±16.5	44.7±10.6
2-AA	127.0±18.2	210.3±53.3	1736.0±138.8	1560.0±52.5	77.3±8.0
NC = Negative Control (untreated) SC = Solvent Control (DMSO) NaN3: sodium acide; 9-AA: 9-aminoacridine; 2-NF: 2-nitrofluorene; MMS: methyl methanesulphonate; 2-AA: 2-aminoanthracene

 

Table 2: Test Results of Experiment 2

EXPERIMENT 2 (pre-incubation method)
S9-Mix	Without
Test item (mg/plate)	TA 1535	TA 1537	TA 98	TA 100	WP2 uvr A
NC	12.0±3.0	17.7±2.1	20.7±8.6	229.3±32.3	22.0±2.6
SC	11.7±2.5	15.7±2.1	15.3±7.8	199.0±39.0	27.7±4.6
0.01	10.3±5.7	6.0±1.7	14.7±3.5	225.3±9.6	24.0±5.0
0.05	8.7±0.6	7.7±3.2	17.7±6.5	201.0±36.6	18.0±4.0
0.1	10.0±1.0	6.3±1.2	25.0±1.7	218.0±28.0	20.3±7.0
0.5	11.0±4.4	3.3±1.5	14.3±3.8	185.3±21.1	22.0±4.0
1	10.7±2.1	2.7±1.5	15.3±5.1	203.3±10.0	23.7±5.7
NaN3	1712.0±40.0			1400.0±172.1
9-AA		1164.0±1.4
2-NF			1400.0±172.1
MMS					256.3±37.7
S9-Mix	With
Test item (mg/plate)	TA 1535	TA 1537	TA 98	TA 100	WP2 uvr A
NC	14.7±9.2	7.3±0.6	46.3±6.0	214.3±9.0	15.3±4.5
SC	14.0±3.5	7.3±4.5	47.3±3.1	216.7±25.2	20.7±1.5
0.01	14.0±3.0	8.3±1.2	44.0±5.6	206.3±25.5	17.0±3.6
0.05	13.7±5.5	11.7±4.2	49.7±8.4	199.3±15.6	16.7±5.1
0.1	12.3±4.9	4.7±0.6	39.7±9.0	193.0±14.0	22.3±4.5
0.5	14.7±3.2	6.3±1.5	41.3±2.9	188.3±4.7	19.0±4.0
1	14.3±2.5	4.7±1.5	32.7±3.8	180.3±6.7	13.3±1.2
2-AA	118.3±14.4	188.3±7.8	1501.3±287.4	1464.0±68.4	206.3±29.7
NC = Negative Control (untreated) SC = Solvent Control (DMSO)  NaN3: sodium acide; 9-AA: 9-aminoacridine; 2-NF: 2-nitrofluorene; MMS: methyl methanesulphonate; 2-AA: 2-aminoanthracene

 

Applicant's summary and conclusion

Conclusions:

Under the conditions of the Ames test the test substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvr A) tested with and without metabolic activation.

Executive summary:

A bacterial gene mutation assay (2001) with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP. In two independent experiments, the S. typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and the E. coli strain WP2 uvr A were exposed to the test substance using the plate incorporation method (experiment 1) and pre-incubation method (experiment 2). In a toxicity range-finding study, in which 6 concentrations up to 5000 µg/plate were tested in TA 100 and E. coli WP2 uvr A, precipitation of the test substance, which interfered with scoring, was observed at 5000 µg/plate. Therefore concentrations of 10, 50, 100, 500 and 1000 µg/plate were selected for the main experiments. The test substance showed no bacterial toxicity at any dose in both experiments with or without metabolic activation. No biologically relevant increase in revertant numbers was observed after treatment with the test substance in any bacterial strain and at any concentration tested in the presence and absence of metabolic activation. The revertant frequencies of the vehicle control were within the expected range and the positive control chemicals induced marked increases in revertant colonies, demonstrating the effective performance of the experiments. Under the conditions of this experiment, the test substance is considered non-mutagenic in the selected S. typhimurium and E. coli strains in the presence and absence of metabolic activation.