Dodecanal

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2007-02-06 to 2007-03-01

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

A valid study is available for the analogue substance, Undec-10-enal. It is a GLP compliant study conducted in compliance with agreed protocols, with no or minor deviations for the standard test. The read-across is considered to be suitable based on the structural and “mechanistic action” similarities between the target substance (Dodecanal) and source substance (Undec-10-enal) and their similar physico-chemical properties.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

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:

Undec-10-enal

EC Number:

203-973-1

EC Name:

Undec-10-enal

Cas Number:

112-45-8

IUPAC Name:

undec-10-enal

Constituent 2

Reference substance name:

10-undecanal

IUPAC Name:

10-undecanal

Details on test material:

- Name of test material (as cited in study report): 10-undecanal
- Substance type: Colourless to yellowish liquid
- Physical state: Liquid
- Analytical purity: 97.7 % (GC)
- Impurities (identity and concentrations): No data
- Composition of test material, percentage of components: No data
- Isomers composition: No data
- Purity test date: No data
- Batch No: 7500118249
- Expiration date of the lot/batch: 2007-11-10
- Stability under test conditions: No data
- Storage condition of test material: In a refrigerator at 2 °C to 8 °C.

Method

Target gene:

Histidine (his)

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 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Pre-experiment, Experiment I: 3, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate.
Pre-experiment, Experiment Ia TA1535 -S9 mix: 3, 10, 33, 100, 333, 1000 µg/plate.
Experiment II: 3, 10, 33, 100, 333, 1000 µg/plate.

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: Chosen because of its solubility properties and its relative non-toxicity to bacteria (Maron et al. 1981)

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Remarks:

Negative and solvent controls were run concurrently.

Controls 2

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Strains TA 1535, TA100, only, both without metabolic activation.

Controls 3

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Strains TA 1537, TA98 only, both without metabolic activation.

Controls 4

Positive control substance:

methylmethanesulfonate

Remarks:

Strain TA102 only, without metabolic activation.

Controls 5

Positive control substance:

other:

Remarks:

Strains TA1535, TA1537, TA98, TA100, TA102, with metabolic activation.

Controls 6

Remarks:

The stability of the positive control substances in solution was unknown but a mutagenic response in the expected range was considered sufficient evidence of biological stability.

Details on test system and experimental conditions:

METHODS OF APPLICATION:
- Experiments I and Ia: in agar (plate incorporation)
- Experiment II: Preincubation

STRAINS
- Strains were stored as stock cultures in ampules with nutrient broth + 5 % DMSO in liquid nitrogen.

PRECULTURES

- From the thawed ampoules of the strains 0.5 mL of bacterial suspension was transferred into 250 mL Erlenmayer flasks containing 20 mL nutrient medium. A solution of 20 µL ampicillin (25 µg/mL) was added to strains TA98, TA100, TA102. Additionally, 20 µL tetracyline (2 µg/mL) was added to strain TA102. This nutrient medium contained per litre:
-- 8 g Merck nutrient broth (Merck, D-64293 Darmstadt, Germany)
-- 5 g NaCl (Merck, D-64293 Darmstadt, Germany)
- The bacterial cultures were incubated in a shaking water bath for 4 hrs at 37 °C.

- Selective agar plates obtained from Merck, D-64293, Darmstadt, Germany.

- Overlay agar contains per litre:
-- 6.0 g MERCK Agar agar
-- 6.0 g NaCl
-- 10.5 mg L-histidine x HCl x H₂O
-- 12.2 mg Biotin

- Sterilisations were performed at 121 °C in an autoclave.

S9 MIX
Preparation
- Phenobarbital/β-naphthoflavone induced rat liver S9 was used as the metabolic activation system.
- S9 was prepared from 8-12 wk old male Wistar Hanlbm rats, weight approx. 220 - 320 g induced by applications of 80 mg/kg bw phenobarbital i.p. (Desitin, D-22335 Hamburg, Germany) and β-naphthoflavone p.o. (Aldrich, D-89555 Steinheim), each on 3 consecutive days. The livers were prepared 24 hrs after the last treatment.
- The S9 fractions were produced by dilution of the liver homogenate with KCl solution (1 + 3) followed by centrifugation at 9000 g.
- Aliquots of the supernatant were frozen and stored in ampoules at -80 °C. Small numbers of the ampoules can be kept at -20 °C for up to 1 wk.
- Each batch of S9 was routinely tested with 2-aminoanthracene and benzo(a)pyrene.

- The protein concentration in the S9 mix was 38.1 mg/mL.

Utilisation
- Before the experiment an appropriate amount of S9 mix supernatant was thawed and mixed with S9 cofactor solution. The volume of supernatant was 10 % v/v in the S9 mix. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix:
-- 8 mM MgCl₂
-- 33 mM KCl
-- 5 mM glucose-6-phosphate
-- 5 mM NADP
- in 100 mM sodium orthophosphate buffer, pH 7.4

During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al. (1977).

RANGE-FINDING/SCREENING STUDIES
- To evaluate the toxicity of the test item, a pre-experiment was performed with all strains used. 8 concentrations, 3 - 5000 µg/plate were tested for toxity and mutation induction.
- Toxicity of the test item could be evident as a reduction in the number of spontaneous revertants or a clearing of the background lawn.
- The pre-experiment was reported as main Experiment I, since the following criterion was met:
-- Evaluable plates (> 0 colonies) at ≥ 5 concs.

DOSE SELECTION
- Pre-experiment was reported as Experiment 1. Featured concentrations of 3 - 5000 µg/plate.
- Strain TA1535 -S9 mix had to be repeated because only 4 concs were evaluable.
- Based on the toxic effects observed in Experiment I, a lower conc. was chosen for Experiments II and Ia with 1000 µg/plate as maximal conc.

EXPERIMENTAL PROCEDURE
- For each strain and dose level, including the controls, 3 plates were used.
- The following materials were mixed in a test tube and poured onto the selective agar plates:
-- 100 µL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
-- 500 µL S9 mix or S9 mix substitution buffer, as appropriate.
-- 100 µL bacteria suspension
-- 200 µL Overlay agar.
- In the preincubation assay, 100 µL test solution, 500 µL S9 mix/S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and incubated at 37 °C for 60 mins. After preincubation, 2.0 mL overlay agar (45 °C) was added to each test tube. The mixture was poured on minimal agar plates.
- After solidification the plates were incubated upside down for ≥ 48 hr at 37 °C in the dark.

COLONY COUNTING
- Colonies were counted using Petri viewer Mk 2 (perceptive Instruments Ltd, Suffolk, CB9 7BN, United Kingdom) with software Ames Study Manager. The counter was connected to a computer to print out the individual and mean values of the plates for each concentration along with standard deviations and enhancement factors as compared to the spontaneous reversion rates.
- Due to reduced background growth, the colonies were partly counted manuallly.

Evaluation criteria:

A test item was considered mutagenic if biological relevant increase in the number of revertants exceeding the threshold of twice (strains TA98, TA100, TA102) or thrice (strains TA1535, TA1537) the colony count of the corresponding solvent control is observed.

A dose-dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose-dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative and solvent controls, such an increase was not considered biologically relevant.

Statistics:

None; according to OECD guideline 471, statistical analysis of the data is not mandatory.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Please refer to "attached background material" below for full results.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Additional information on cytotoxicity

Reduced background growth was observed at the following concentrations (µg/plate):

Strain	Pre-experiment/Experiment I		Experiment II
	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix
TA1535	100 - 5000100 -1000 (VV a)	333 - 5000	100 - 1000	100 - 1000
TA1537	100 - 5000	333 - 5000	100 - 1000	100 - 1000
TA98	100 - 5000	333 - 5000	100 - 1000	100 - 1000
TA100	100 - 5000	333 - 5000	33 - 1000	100 - 1000
TA102	100 - 5000	333 - 5000	100 - 1000	100 - 1000

Distinct toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5 were observed at the following concentrations (µg/plate):

Strain	Pre-experiment/Experiment I		Experiment II
	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix
TA1535	333 - 5000333 - 1000 (VV a)	1000 - 5000	333 - 1000	333 - 1000
TA1537	333 - 5000	1000 - 5000	333 - 1000	333 - 1000
TA98	333 - 5000	1000 - 5000	100 - 1000	100 - 1000
TA100	333 - 5000	1000 - 5000	33 - 1000	1000
TA102	333-5000	1000 - 5000	333 - 1000	1000

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test substance was assessed for genotoxicity according to OECD guideline 471 with and without metabolic activation. Under the conditiosn of the test, the test substance was not mutagenic either with or without metabolic activation.
A valid study is available for the analogue substance, Undec-10-enal. It is a GLP compliant study conducted in compliance with agreed protocols, with no or minor deviations for the standard test. The read-across is considered to be suitable based on the structural and “mechanistic action” similarities between the target substance (Dodecanal) and source substance (Undec-10-enal) and their similar physico-chemical properties.