sec-butylamine

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:

03 November 2016 - 15 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

28 July 2015.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

30 May 2008.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

sec-butylamine

EC Number:

237-732-7

EC Name:

sec-butylamine

Cas Number:

13952-84-6

Molecular formula:

C4H11N

IUPAC Name:

butan-2-amine

Test material form:

liquid

Method

Target gene:

Thymidine Kinase

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium
pyruvate (200 µg/mL). This medium was supplemented by heat inactivated horse serum at 10% (v/v) or 20% (v/v).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yesCELLS USED
- Source of cells:
- Suitability of cells:
- Cell cycle length, doubling time or proliferation index:
- Sex, age and number of blood donors if applicable:
- Whether whole blood or separated lymphocytes were used if applicable:
- Number of passages if applicable:
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes:
- Normal (negative control) cell cycle time:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
- Properly maintained: [yes/no]
- Periodically checked for Mycoplasma contamination: [yes/no]
- Periodically checked for karyotype stability: [yes/no)
- Periodically 'cleansed' against high spontaneous background: [yes/no]

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

n/a

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

Since the test item was found freely soluble but cytotoxic in the preliminary test, the highest dose level for the main experiments was selected on the basis of the cytotoxicity, according to the criteria specified in the international guidelines.

Experiments without S9 mix:
- 50, 100, 200, 400, 600, 800, 1000 and 2000 µg/mL for the first experiment,
- 200, 400, 600, 800, 1000 and 2000 µg/mL for the second experiment.

Experiments with S9 mix:
- 50, 100, 200, 400, 600, 1000, 1500 and 2000 µg/mL for the first experiment,
- 200, 400, 600, 1000, 1166.7, 1333.3 and 1500 µg/mL for the second experiment.

Vehicle / solvent:

- Vehicle used: water for injections
- Justification for choice: according to available solubility data, test item was soluble in the vehicle at 400 mg/mL.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: methylmethanesulfonate (-S9 mix); cyclophosphamide (+S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11-12 days

SELECTION AGENT (mutation assays): trifluorothymidine

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth.

NUMBER OF REPLICATIONS: one culture/dose elevl for the preliminary test; two cultures/dose level for the main experiments.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth.

Evaluation criteria:

IWGT recommendations were followed for the determination of a positive result, which should fulfill both following criteria:
- at least at one dose level the mutation frequency minus the mutation frequency of the vehicle control (IMF) equals or exceeds the Global Evaluation Factor (GEF) of 126 x 10-6,
- a dose-response relationship is demonstrated by a statistically significant trend test.

Unless an effect is considered as clearly positive, the reproducibility of a positive effect should be confirmed.

Noteworthy increases in the mutation frequency observed only at high-levels of cytotoxicity (Adj. RTG lower than 10%), but with no evidence of mutagenicity at dose levels with Adj. RTG between 10 and 20%, are not considered as positive results.
A test item may be considered as non-mutagenic when there is no culture showing an Adj. RTG value between 10 and 20% if:
- there is at least one negative data point between 20 and 25% Adj. RTG and no evidence of mutagenicity in a series of data points between 100 and 20% Adj. RTG,
- there is no evidence of mutagenicity in a series of data points between 100 and 25% and there is also a negative data point between 10 and 1% Adj. RTG.

Statistics:

To assess the dose-response relationship, a linear regression was performed between dose levels and individual mutation frequencies obtained at dose levels showing a mean Adj. RTG >= 10%. This statistical analysis was performed using SAS Enterprise Guide software.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without S9 mix: marked to severe cytotoxicity at dose levels >= 1000 µg/mL. With S9 mix: - First experiment: slight to severe cytotoxicity at dose levels >= 600 µg/mL. - Second experiment: moderate to severe cytotoxicity at dose levels >= 1000 µg/mL.

Vehicle controls validity:

valid

Remarks:

Without S9 mix: CE2 below the range [0.65-1.2]. However, all other acceptance criteria met for the vehicle control, and positive control response met both criteria, demonstrating the sensitivity of the test system. Overall results thus considered valid.

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:pH shift was observed in the culture medium during the preliminary cytotoxicity test. When compared to the corresponding vehicle control medium, pH shifts of more than one unit were noted in the first experiment at dose levels >= 1500 µg/mL, and in the second experiment at dose levels >= 600 µg/mL.
- Effects of osmolality: none.
- Precipitation:

RANGE-FINDING STUDIES: Using a test item concentration of 400 mg/mL and a treatment volume of 0.5% (v/v) in the culture medium (i.e. 100 µL/20 mL culture medium), the highest recommended dose level of 2000 µg/mL was achievable. Thus, the dose levels selected for the treatments of the preliminary test were 4, 40, 200, 400, 1000 and 2000 µg/mL.

At the three highest tested dose levels of 400, 1000 and 2000 µg/mL, the pH of the culture medium was approximately 6.8, 7.4 and 9.1, respectively (6.8 for the vehicle control), and the osmolality of the highest tested dose level of 2000 µg/mL was 326 mOsm/kg H2O (307 mOsm/kg H2O for the vehicle control).
Care should be taken when interpreting the results at dose levels > 1000 µg/mL (particularly in order to avoid false positive results), since the pH shift observed at 2000 µg/mL relative to the vehicle control culture was of more than one unit.

At the end of the treatment periods, neither precipitate nor emulsion was observed in the culture medium at any of the tested dose levels.
A pink coloration of the culture medium was recorded at dose levels >= 1000 µg/mL, which may be related to the pH increase and to the cytotoxicity observed at these dose levels.

Following the 3-hour treatment without S9 mix, a severe cytotoxicity was induced at dose levels >= 1000 µg/mL, as shown by a 92 to 100% decrease in Adj. RTG.
Following the 3-hour treatment with S9 mix, a slight to severe cytotoxicity was induced at 40 µg/mL and at dose levels >= 400 µg/mL, as shown by a 43 to 100% decrease in Adj. RTG.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): see document attached.

Applicant's summary and conclusion

Conclusions:

The test item did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.

Executive summary:

The objective of this study was to evaluate the potential of the test item to induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK^+/- mouse lymphoma cells.

 

The study was performed according to the international guidelines and in compliance with the principles of Good Laboratory Practice.

 

Methods

After a preliminary cytotoxicity test, the test item, prepared in water for injections, was tested in two independent experiments, with and without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Cultures of 20 mL at 5 x 10⁵cells/mL were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% in a 37°C, 5% CO₂ humidified incubator.

 

Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE₂).

The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.

 

Results

With one exception which was considered not to have compromised the integrity or validity of the study, the cloning efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria.

For the positive control cultures, the increase in the mutation frequencies met also the acceptance criteria.In addition, the upper limit of cytotoxicity observed in the positive control cultures had an Adj. RTG greater than 10%. The study was therefore considered to be valid.

 

Since the test item was found cytotoxic and freely soluble in the preliminary test, the highest dose level for the main experiments was selected on the basis of the cytotoxicity, according to the criteria specified in the international guidelines.

 

Neither precipitate nor emulsion was observed in the culture medium at any dose levels, at the end of the treatment period.

 

Since a pH shift was observed in the culture medium during the preliminary cytotoxicity test, the pH values were also recorded in the main experiments for all or part of the tested dose levels, and for the vehicle control cultures. pH shifts of more than one pH unit when compared to the corresponding vehicle control medium were noted at dose levels >= 1500 µg/mL in the first experiment, and at dose levels >= 600 µg/mL in the second experiment.

 

Experiments without S9 mix

The selected dose levels were as follows:

.            50, 100, 200, 400, 600, 800, 1000 and 2000 µg/mL for the first experiment,

.            200, 400, 600, 800, 1000 and 2000 µg/mL for the second experiment.

Cytotoxicity

A marked to severe cytotoxicity was induced at dose levels >= 1000 µg/mL, as shown by a 81 to 100% decrease in Adj. RTG.

 

Mutagenicity

In the first experiment, a slight but dose-related increase in the MF was observed at dose levelsinducing acceptable levels of cytotoxicity, i.e. up to 1000 µg/mL (p < 0.05). Sincenone of the induced mutation frequencies exceeded the GEF of +126 x 10^-6, a second experiment was conducted in order to check the reproducibility of the first results obtained.

Similar results were then obtained in the second experiment, since a slight but dose-related increase in the MF was observed at dose levels inducing acceptable levels of cytotoxicity, i.e. up to 1000 µg/mL (p < 0.05). As for the first experiment, none of the IMF exceeded the GEF.

 

Since IMF values obtained in both experiments remained substantially below the GEF, despite the narrow ranges of dose levels used, the linear trends were considered as meaningless and the overall results without S9 mix were considered to meet the criteria of a negative response.

 

Experiments with S9 mix

The selected dose levels were as follows:

.            50, 100, 200, 400, 600, 1000, 1500 and 2000 µg/mL for the first experiment,

.            200, 400, 600, 1000, 1166.7, 1333.3 and 1500 µg/mL for the second experiment.

 

Cytotoxicity

In the first experiment, a slight to severe cytotoxicity was induced at dose levels >= 600 µg/mL, as shown by a 50 to 100%decrease in Adj. RTG.

In the second experiment, a moderate to severe cytotoxicity was induced at dose levels >= 1000 µg/mL, as shown by a 66 to 100%decrease in Adj. RTG.

 

Mutagenicity

In the first experiment,no or only slight increase in the MF, remaining substantially below the GEF, were observed at dose levels inducing acceptable levels of cytotoxicity (i.e. up to 1000 µg/mL), and no dose-response relationship was demonstrated by the linear regression (p > 0.05).

An increase in the mutation frequency, exceeding the GEF, was observed at the dose level of 1500 µg/mL (IMF value of 235), however this increase was associated with a high-level of cytotoxicity (Adj. RTG of 2%), and was thus considered as not relevant in terms of mutagenicity.

Since none of the tested dose levels induced the recommended level of cytotoxicity (i.e. Adj. RTG between 10 and 20%), and since there was no negative data points between 1 and 10% Adj. RTG, a second experiment was conducted using a narrower range of dose levels.

 

In the second experiment, only slight increases in the MF, remaining substantially below the GEF, were observed at dose levels inducing acceptable levels of cytotoxicity (i.e. up to 1000 µg/mL), but a dose-response relationship was demonstrated by the linear regression (p < 0.01).

None of the tested dose levels induced the recommended level of cytotoxicity (i.e. Adj. RTG between 10 and 20%), however,there is no evidence of mutagenicity in a series of data points between 100 and 25% and there is also a negative data point between 10 and 1% Adj. RTG.

 

Since the MF values remained substantially below the GEF (maximal IMF value of 44 x 10 ^-6), the linear trend was considered as meaningless and the overall results with S9 mix were considered to meet the criteria of a negative response.

Conclusion

Under the experimental conditions of this study, the test item did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.