Butanamide, N-(3-ethoxypropyl)-2,4-dihydroxy-3,3-dimethyl-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames test, a chromosome aberration test and a HPRT (chinese hamster V79 cells) were conducted. The studies show no genotoxic/mutagenic or clastogenic effects of the test item.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-08-12 to 2008-10-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

The V79 cells are exposed to the test item both with and without exogenous metabolic activation. At a defined time interval after treatment the descendants of the treated orginal population are monitored for the loss of functional HPRT enzyme.
HPRT (hypoxanthine- guanine phosphoribosyl transferase) catalyes the conversion of the nontoxic 6TG (6-thioguanine) to its toxic ribophosphorylated derivate. Therefore, cells defient HPRT due to a forward mutation are resistant to 6TG. These cells are able to proliferate in the presence of 6TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 - 9 days. The expression period is terminated by adding 6Tg to culture medium.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: HAT-medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/ß-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

150; 300; 600; 1200; 2400 µg/mL

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relative non-toxicity to the cells.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Migrated to IUCLID6: without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

Migrated to IUCLID6: With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; MEM supplemented with 10 % FCS,

DURATION
- Exposure duration: First experiment: 4 hours; second experiment: 24 hours
- Expression time: 7 days
- Selection time: 8 days

SELECTION AGENT: 6-TG

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 1.5 x 10E06

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency;

OTHER EXAMINATIONS:
- Osmolarity: solvent control. 393 mOsm; test item/2400 µg/mL: 381
- pH: solvent control: 7.3; test item/2400 µg/mL: 7.3

OTHER: none

Evaluation criteria:

A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutgenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concentrations a mutation frequency that is three times higher than the spontaneous mutation frequency in the experiment.
The test item is classfied as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutatant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data.

Statistics:

A linear regression was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 statistics software. The number of mutant colonies obtained for the groups treated with test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value is below 0.05. However, both, biological and statistical significance were considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: no
- Water solubility: soluble
- Precipitation: no precipitation of the testitem up to the maximal concentration in all experiments.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: A pre-test was performed according to the guidelines. Test item concentrations were chosen with regard to the purity of the test item (100.3 %) and its molecular weight (233.31 g/mol).

COMPARISON WITH HISTORICAL CONTROL DATA: no

ADDITIONAL INFORMATION ON CYTOTOXICITY: no

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Executive summary:

In a mammalian cell gene mutation assay (1197800), in chinese hamster V79 cells cultured in vitro were exposed for 4 hours to the test item, at concentrations of 150, 300, 600, 1200, 2400 µg/mL in the presence and absence of mammalian metabolic activation S9 mix (rat liver). In a second test the chinese hamster V79 cell cultures were exposed to the same concentrations for 24 hours in the absence of metabolic activation. The test item was tested up to concentrations of 2400 µg/mL (approx 10 mM). No relevant cytotoxic effect was observed in the first experiment as relative cloning efficiency 1 did not go below 50 %. In the second experiment cytotoxicity was noted at 300 µg/mL and above. No substantial dose dependent increase of the mutation frequency exceeding the threshold of three times the mutation frequency of the corresponding solvent control occured with and without metabolic activation. Furthermore there was no dose dependent trend even below the threshold mentioned above as indicated by the missing statistical significance. Therefore, the data of this study are judged as non-mutagenic.  The positive controls induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test and the activity of the metabolic activation system.   This study is classified as acceptable.  This study fulfills the requirements of the Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The test item was evaluated for genotoxic activity in two versions of the Ames test: the standard plate incorporation and the pre-incubation methods. TheS. typhimuriumstrains TA 98, TA 100, TA1535, TA 1537, TA1538 and the E. Colistrain WP2uvrA were used in the study. Metabolic activation was provided by S9- mix derived from the livers of Aroclor 1254 pre-treated rats.

The bacterial cells were exposed to the test item up to the generally recommended upper dose limit of 5000 µg/plate. No precipitate and no toxic effects were apparent up to this concentration. The test item did not cause any increase of the number of revertant colonies in any of the six tested strains while the concurrently tested positive controls documented the responsiveness of the strains and the activity of the metabolic activation system.

It is therefore concluded that the test item is not genotoxic in the Ames test under the described experimental conditions.

The test item was assessed as to its ability to induce chromosomal aberrations in human peripheral blood lymphocytes in vitro. Without metabolic activation doses between 333 and 5000 µg/mL were tested after 24 hours continuous treatment. With metabolic activation (S9- mix, rat) doses between 1000 and 5000 µg/mL were tested after a 3 hours pulse treatment. Two independent experiments were performed at a fixation period of 24 hours. Additionally the highest dose of 5000 µg/mL was tested in one experiment at a fixation period of 48 hours (i. e. after a 48 h continuous treatment in absence and a 3 hours pulse treatment in presence of S9- mix).

The sensitivity of the test system and the activity of the metabolic activation were demonstrated by using the direct acting mutagen mitomycin-C (MMC-C) and the promutagen cyclophosphamide (CP) as positive controls. Both substances increased significantly the rate of chromosome aberrations.

The highest dose assayed was the maximal recommended one. Cytotoxicity as measured by reductions in the mitotic indices (MI) was observed after continuous (24 and 48 hours) exposures to the test item in both experiments. Exposure to the test item did not raise the rate of cells with chromosome aberrations.

In a mammalian cell gene mutation assay, in chinese hamster V79 cells cultured in vitro were exposed for 4 hours to the test item, at concentrations of 150, 300, 600, 1200, 2400 µg/mL in the presence and absence of mammalian metabolic activation S9 mix (rat liver). In a second test the chinese hamster V79 cell cultures were exposed to the same concentrations for 24 hours in the absence of metabolic activation.

No relevant cytotoxic effect was observed in the first experiment as relative cloning efficiency 1 did not go below 50 %. In the second experiment cytotoxicity was noted at 300 µg/mL and above. No substantial dose dependent increase of the mutation frequency exceeding the threshold of three times the mutation frequency of the corresponding solvent control occurred with and without metabolic activation. Furthermore there was no dose dependent trend even below the threshold mentioned above as indicated by the missing statistical significance. Therefore, the test item is judged as non-mutagenic.  

 The studies discussed above show no genotoxic/mutagenic or clastogenic effects of the test item.

Justification for selection of genetic toxicity endpoint
GLP and guideline study on mammalian cells

Justification for classification or non-classification

Based on the results obtained in in vitro studies the test item is not considered to be genotoxic/mutagenic or clastogenic and thus has not to be classified mutagenic according to Council Directive 67/548/EEC and Regulation (EC) No 1272/2008.