Decanal

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1980-10-13 to 1980-12-15

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study meets generally accepted scientific standards with acceptable restrictions for the standard test.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1981

Report date:

1981

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: Clive D, Spector JFS (1975). Laboratory procedures for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells. Mutation Research Vol 31 pp 17-29.

Principles of method if other than guideline:

Mouse lymphoma assay

GLP compliance:

no

Type of assay:

mammalian cell gene 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

Details on test material:

- Name of test material (as cited in study report): Nonanal
- Molecular formula: C₉H₁₈O
- Molecular weight: 397.09
- Smiles notation: CCCCCCCCC=O
- InChl: InChI=1S/C9H18O/c1-2-3-4-5-6-7-8-9-10/h9H,2-8H2,1H3
- Structural formula attached as image file.
- Appearance: Clear liquid
- Lot No.: 2F4107
- Expiration date of the lot/batch: 2003-03
- Storage condition of test material: room temperature in the dark

Specific details on test material used for the study:

- Name of test material (as cited in study report): C9 aldehyde, C-192 (SN 2188-2)
- Molecular weight (if other than submission substance): 142.24
- Smiles notation (if other than submission substance): O=CCCCCCCCC
- Substance type: Clear, colourless liquid
- Physical state: Liquid
- Analytical purity: No data
- Impurities (identity and concentrations): No data
- Composition of test material, percentage of components: No data
- Isomers composition: No data
- Purity test date: No data
- Lot/batch No: No data
- Expiration date of the lot/batch: No data
- Stability under test conditions: No data
- Storage condition of test material: Room temperature under a blanket of nitrogen to avoid deterioration due to oxidation.

Method

Target gene:

TK

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Rat S-9 liver homogenate

Test concentrations with justification for top dose:

Without metabolic activation: 0 (control), 0.0977 nL/mL, 0.195 nL/mL, 6.25 nL/mL, 12.5 nL/mL, 25 nL/mL
With metabolic activation: 0 (control), 0.0977 nL/mL, 0.195 nL/mL, 6.25 nL/mL, 12.5 nL/mL, 25 nL/mL, 50 nL/mL, 60 nL/mL, 75 nL/mL, 100 nL/mL, 120 nL/mL.

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Was immiscible in water at 100 µL/mL but dissolved in DMSO at that concentration.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Controls 2

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Concentration: 0.5 µL/L; without metabolic activation only

Controls 3

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

Concentration: 0.3 µL/L; with metabolic activation only

Details on test system and experimental conditions:

INDICATOR CELLS
- Mouse lymphoma cell line was derived from the Fischer L5178Y line of Dr Donald Clive.
- Stocks were maintained in liquid nitrogen and laboratory cultures were periodically checked for the absence of mycoplasma contamination by culturing methods
- To reduce the negative control frequency (spontaneous frequency) of TK-/- mutants to as low as possible, cell cultures were exposed to conditions that selected against the TK-/- phenotype (exposure to methotrexate) and are then returned to normal growth medium for ≥ 3 d before use.

MEDIA
- Cells were maintained in Fischer's mouse leukaemia medium supplemented with L-glutamine, sodium pyruvate and horse serum (10 % by volume).
- Cloning medium consisted of the preceding growth medium with the addition of agar to a final concentration of 0.35 % to achieve a semi-solid state.
- Selection of medium is cloning medium containing 100 µg/mL of BrdU or 3 µg/mL of TFT.

DOSE SELECTION
- A wide range of chemical concentrations was tested for cytotoxicity, starting with a maximum applied dose of 1 mg/mL for solutions in organic solvents.
- After a 4 hr exposure, the cells were washed and a viable count was obtained the next day.
- Relative cytotoxicities expressed as the reduction in growth compared to the growth of untreated cells was used to select 7 to 10 doses that cover the range from 0% to 50-90 % reduction in 24 hr. These selected doses were subsequently applied to cell cultures prepared for mutagenicity testing, but only 4 or 5 of the doses would be carried through to the mutant selection process. This procedure compensated for daily variations in cellular cytotoxicity and ensured that the choice of 4 or 5 doses spaced from 0 to 50- 90 % reduction in cell growth.

MUTAGENICITY TESTING
- Cultures exposed to the test chemical for 4 hrs at the pre-selected doses were washed and placed in growth medium for 2-3 d to allow recovery, gworth and expression of the TK-/- genotype. Cell counts were determined daily and appropriate dilutions were made to allow optimal growth rates.
- At the end of the expression period, 3 × 10⁶ cells for each selected dose were seeded in soft agar plates with selection medium and resistant (mutant) colonies were counted after 10 d incubation. To determine the actual number of cells capable of forming colonies, a portion of the cell suspension was also cloned in a normal, non-selective, medium. The ratio of resistant colonies to total viable cell number is the mutant frequency.
- A detailed flow diagram of the mutation assay is attached under supporting information below.
- The non-activation assay was run as described as above.
- The activation assay was run concurrently with the non-activation assay. The only difference is the addition of the S-9 fraction of rat liver homogenate and necessary cofactors (CORE) during the 4 hr treatment period. CORE consists of NADP (sodium salt) and isocitric acid. The final concentrations of the activation system component in the cell suspension were: 2.4 mg NADP/mL, 4.5 mg isocitric acid/mL and 50 µg S-9/mL.

PREPARATION OF S-9 LIVER HOMOGENATE
- A 9000 × g supernatant prepared from Fischer 344 adult male rat liver induced by Aroclor 1254 (described by Ames et al. 1975) was purchased from Bionetics Laboratory Products, Litton Bionetics, Inc.

Evaluation criteria:

A test is considered positive (mutagenic) if:
- The mutation frequency exceeds 150 % of the concurrent background frequency by ≥ 10⁻⁴, and:
-- A dose-related or toxicity-related increase in mutant frequency, preferably in ≥ 3 doses.
-- Or, if an increase of ~ 2 × the minimum criterion or greater is observed for a single dose near the highest testable toxicity.
-- However, if an increase in mutant frequency is followed by only small or no further increase in toxicity, and there is a decrease in mutant frequency to values below the minimum criterion is not acceptable to classify the substance as mutagenic.
-- Either parameter, applied concentration or toxicity can be used to establish whether the mutagenic activity is related to an increase in effective treatment. A negative correlation with dose is acceptable only if a positive correlation with toxicity exists. An apparent increase in mutagenic activity as a function of decreasing toxicity is not acceptable evidence for mutagenicity.

A test is considered negative (non-mutagenic) if:
- If the minimum increase in frequency is not observed for a range of applied concentrations that extends to toxicity causing 5 % to 10 % relative suspension growth.

Statistics:

None

Results and discussion

Test results

Test results 1

Species / strain:

mouse lymphoma L5178Y cells

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:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

tested up to highest dose of 50 nL/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Appeared to be soluble up to 125 nL/mL but cloudiness that was indicative of immiscible droplets was observed from 250 nL/mL to 1000 nL/mL.

RANGE-FINDING/SCREENING STUDIES:
- 24 hr growth was severely reduced by exposure to 31 nL/mL and treatments with 62.5 nL/mL and higher were completely lethal. Therefore, the mutation assay was initiated with a series of concentrations from 50 nL/mL to 0.0977 nL/mL in 2-fold dilution steps to cover a wide range of toxic action.

NON-ACTIVATION CONDITIONS
- A single assay was performed under non-activation conditions, the results of which are given in table 1 in the attached supporting information.
- Under non-activation conditions, the mutant frequencies in the cultures remained strictly comparable to the negative (solvent and untreated) controls. The assayed treatments caused weak to moderate toxicities that resulted in a relative growth range of 69.8 % to 21.8 %. The toxicity was not closely related to the applied concentration, although the 25 nL/mL was the most toxic. Treatment with 50 nL/mL was completely toxic to the cells. A concentration approaching excessive lethality was closely approached without there obtaining any evidence of mutagenic activity. The test material was therefore evaluated as non-mutagenic under these conditions.

ACTIVATION CONDITIONS
- Three assays were performed under activation conditions, the details of which are given in tables 2-4 in the attached supporting information.
- In the first assay with metabolic activation (table 2), concentrations up to 120 nL/mL were examined. The exposure to 120 nL/mL was highly toxic (6.1 % relative growth), and treatment with 150 nL/mL was completely lethal. Increases in mutant frequency were observed for the 100 nL/mL and 120 nL/mL treatments that appeared to indicate weak mutagenic activity. However, the very low negative control values mutant frequencies introduced uncertainty into the calculation of the frequency of 21.9 × 10⁻⁶ as the minimum criterion for indicating mutagenesis. This frequency was just exceeded for the culture exposed to 100 nL/mL and closely approached by the 120 nL/mL treatment. Also, since the DMN positive control induced a mutant frequency just below the usual lower limit of 200 × 10⁻⁶, the activation efficiency of the S-9 mix may have been subnormal. Thus, a larger mutagenic increase might be observed for the test material in another trial where DMN activation was normal. Another trial was also necessary to determine whether the small increases were spurious or could be confirmed.

- In the 2nd assay with metabolic activation (table 3), good activation of DMN was demonstrated. Small increases in mutant frequency were observed for treatments in the 25 to 120 nL/mL range. The minimum criterion for indicating mutagenesis in this trial was a mutant frequency of 46.8 × 10⁻⁶. This value was exceeded in 3 cultures and closely approached in 2 others. The increases tended to correlated with increased frequency; thus the 6.25 nL/mL treatment, which caused 36.4 % relative growth, did not result in a significant change in mutant frequency. Treatment with 25 nL/mL (24.1 % relative growth) induced a 1.9 × increase over background. The four treatments from 60 nL/mL to 120 nL/mL were about equally toxic (10 to 15 % relative growth) and induced a 1.2 × increase. These results indicated a weak mutagenic activity associated with high toxicity and apparently reaching saturation at concentrations below 120 nL/mL. This saturation could be caused by the solubility limit since the presence of fine droplets of test material could be difficult to detect in the cloudy culture media and higher concentrations did result in noticeable cloudiness. The results from trials 2 and 3 were considered confirmatory and indicative of weak mutagenic activity for ≥ 1 product(s) of the test material formed by rat S-9 mix. Based on the new guidance for assessment of mouse lymphoma data, this study is considered to be negative.

- The average cloning efficiencies for the negative controls varied from 86 % without activation to 75 % (table 1) and 82 % (table 3) with activation, which demonstrated good culturing conditions for the assays. The negative control mutant frequencies were normal (but low in trial 2), and the positive control compounds induced mutant frequencies that were generally in excess of the backgrounds.

Remarks on result:

other: strain/cell type: mouse lymphoma L5178Y cells

Applicant's summary and conclusion

Conclusions:

The test substance was assessed for genotoxicity in a mouse lymphoma assay with and without metabolic activation. The results of the test were negative with and without metabolic activation.