sec-butylamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There are three GLP in vitro tests available.

Link to relevant study records

Reference

Reference 1

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

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

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

Target gene:

Thymidine Kinase

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.

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.

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.

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 November 2016 - 16 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

26 September 2014.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable (not a gene mutation assay).

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium containing 10% heat-inactivated horse serum, L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

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 selected for the main experiments was based on the level of cytotoxicity, according to the criteria specified in the international regulations.

Experiments without S9 mix
Using a treatment volume of 0.5% (v/v) in the culture medium, the selected dose levels were as follows:
- 50, 100, 200, 400, 600, 800 and 1000 µg/mL for the first experiment (3- and 24-hour treatments),
- 200, 400, 600, 800, 1000, 1400, 1750 and 2000 µg/mL for the second experiment (3-hour treatment).

Experiments with S9 mix
Using a treatment volume of 0.5% (v/v) in the culture medium, the selected dose levels were 200, 400, 600, 1000, 1400, 1750 and 2000 µg/mL.

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: mitomycin C, colchicine (-S9 mix); cyclophosphamide (+S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium.

DURATION:
Preliminary cytotoxicity test:
Without S9 mix:
3-hour treatment + 24-h recovery
24-hour treatment + 0-h recovery

With S9 mix: 3-hour treatment + 24-h recovery.

Main cytogenetic tests:
First experiment
Without S9 mix:
3-hour treatment + 24-h recovery
24-hour treatment + 0-h recovery

With S9 mix: 3-hour treatment + 24-h recovery.

Second experiment
Without S9 mix: 3-hour treatment + 24-h recovery.

NUMBER OF CELLS EVALUATED: 2000 mononucleated/dose.

DETERMINATION OF CYTOTOXICITY
- Method: population doubling.

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

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After the final cell counting, the cells were washed with culture medium containing 10% inactivated horse serum and 1% pluronic acid. The cells were suspended in 49.5% culture medium containing 10% inactivated horse serum, 50% PBS and 0.5% pluronic acid, before being fixed. Following the fixation, the cells were kept at 4°C for at least an overnight period.

Depending on the observation at the end of the recovery period (presence or absence of precipitate and/or cytotoxicity), three dose levels of the test item-treated cultures were selected for spreading on slides. Cells were dropped onto clean glass slides. The slides were air dried before being stained for approximately 15 min in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above.


CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Analysis was performed under a microscope (1000 x magnification), on the basis of the recommendations of Miller et al. (1995) (e), according to the following criteria:
- micronuclei should be clearly surrounded by a nuclear membrane,
- the micronucleus area should be less than one-third of the area of the main nucleus,
- non-refractility of the micronuclei,
- micronuclei should not be linked to the main nucleus via nucleoplasmic bridges,
- micronuclei should be located within the cytoplasma of the cell,
- only mononucleated cells with a number of micronuclei = 5 should be scored to exclude apoptosis and nuclear fragmentation.

Evaluation criteria:

The biological relevance of the results was always taken into account when evaluating results.

Evaluation of a positive response: a test item is considered to have clastogenic and/or aneugenic potential, if all the following criteria were met:
- a dose-related increase in the frequency of micronucleated cells is demonstrated by a statistically significant trend test,
- for at least one dose level, the frequency of micronucleated cells of each replicate culture is above the corresponding vehicle historical range,
- a statistically significant difference in comparison to the corresponding vehicle control is obtained at one or more dose levels.

Evaluation of a negative response: a test item is considered clearly negative if none of the criteria for a positive response was met.

Statistics:

For each condition of the cytogenetic experiment, the frequency of micronucleated cells in treated cultures was compared to that of the vehicle control cultures. This comparison was performed using the Khi2 test, unless treated culture data are lower than or equal to the vehicle control data. P = 0.05 was used as the lowest level of significance. This statistical analysis was performed using a validated Excel sheet.

To assess the dose-response trend, a linear regression was performed between the frequencies of micronucleated cells and the dose levels. This statistical analysis was performed using SAS Enterprise Guide software.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without S9 mix: - 3-hour treatments: slight to severe cytotoxicity at dose levels >= 800 µg/mL. - 24-hour treatment: marked to severe cytotoxicity at dose levels >= 600 µg/mL. With S9 mix: marked to severe cytotoxicity at dose levels >= 1750 µg/mL.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: a pH shift was observed in the culture medium during the preliminary test.
In the main experiments, pH shifts of more than one unit when compared to the corresponding vehicle control medium were noted at dose levels >= 1750 µg/mL (first experiment) and >= 200 µg/mL (second experiment).

- Effects of osmolality:none.
- Evaporation from medium: none.
- Precipitation: neither precipitate nor emulsion was observed in the culture medium at the end of the treatment period, at any dose levels.
- Definition of acceptable cells for analysis: Analysis was performed under a microscope (1000 x magnification), on the basis of the recommendations of Miller et al. (1995) (e), according to the following criteria:
- micronuclei should be clearly surrounded by a nuclear membrane,
- the micronucleus area should be less than one-third of the area of the main nucleus,
- non-refractility of the micronuclei,
- micronuclei should not be linked to the main nucleus via nucleoplasmic bridges,
- micronuclei should be located within the cytoplasma of the cell,
- only mononucleated cells with a number of micronuclei = 5 should be scored to exclude apoptosis and nuclear fragmentation.

- Other confounding effects: none.

RANGE-FINDING/SCREENING STUDIES:
Using a test item concentration of 400 mg/mL in the vehicle and a treatment volume of 0.5% (v/v) in the culture medium (i.e. 10 µL/2 mL culture medium), the highest recommended dose level of 2000 µg/mL was achievable.
Thus, the dose levels selected for the treatment of the preliminary test were 4, 40, 200, 400, 1000 and 2000 µg/mL.

At the three highest dose levels of 400, 1000 and 2000 µg/mL, the pH of the culture medium was approximately 7.7, 8.2 and 9.1 respectively (7.4 for the vehicle control), and the osmolality of the highest tested dose level of 2000 µg/mL was 328 mOsm/kg H2O (300 mOsm/kg for the vehicle control).
Care should be taken when interpreting the results at 2000 µg/mL (particularly in order to avoid false positive results), since the pH shift observed 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.

Following the 3-hour treatment without S9 mix, a moderate to severe cytotoxicity was observed at dose levels >= 200 µg/mL, as shown by a 41 to 100% decrease in PD.
Following the 24-hour treatment without S9 mix, a severe cytotoxicity was observed at dose levels >= 1000 µg/mL, as shown by a 100% decrease in PD.
Following the 3-hour treatment with S9 mix, a slight to severe cytotoxicity was observed at dose levels >= 200 µg/mL, as shown by a 25 to 100% decrease in PD.

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: see attached document.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see attached document.
- Negative (solvent/vehicle) historical control data: see attached document.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Population Doubling

Conclusions:

The test item, 2-AMINOBUTANE, did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, 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 an increase in the frequency of micronucleated cellsin the mouse cell line L5178Y TK^+/-.

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, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:

 

	First experiment	Second experiment
Without S9 mix	3 h treatment + 24 h recovery 24 h treatment + 0 h recovery	3 h treatment + 24 h recovery None
With S9 mix	3 h treatment + 24 h recovery	None

 

Each treatment was coupled to an assessment of cytotoxicity at the same dose levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells.

Then, after the final cell counting, the cells were washed and fixed. Cells from three dose levels of the test item-treated cultures were then dropped onto clean glass slides. The slides were air-dried before being stained in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above. All slides were coded before analysis, so that the analyst was unaware of the treatment details of the slide under evaluation ("blind" scoring).

For each main experiment (with or without S9 mix), micronuclei were analyzed for three dose levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose). Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.

 

Results

The mean Population Doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.

 

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

 

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

 

Since a pH shift was observed in the culture medium during the preliminary test, pH values were also recorded in the main experiments for all or part of the tested dose levels, and for the vehicle control cultures.

In the main experiments, pH shifts of more than one unit when compared to the corresponding vehicle control medium were noted at dose levels = 1750 µg/mL (first experiment) and = 200 µg/mL (second experiment).

Experiments without S9 mix

The selected dose levels were as follows:

.            50, 100, 200, 400, 600, 800 and 1000 µg/mL for the first experiment (3- and 24-hour treatments),

.            200, 400, 600, 800, 1000, 1400, 1750 and 2000 µg/mL for the second experiment (3-hour treatment).

 

Cytotoxicity

Following the 3-hour treatment of the first experiment, a slight to moderate cytotoxicity was induced at dose levels >= 800 µg/mL, as shown by a 27 to 48% decrease in the PD.

Since the highest tested dose level did not allow reaching the recommended level of cytotoxicity (i.e. 55 ± 5% decrease in the PD), a second experiment was undertaken following a 3-hour treatment and using a revised range of doses.

 

Following the 3-hour treatment of the second experiment, a moderate to severe cytotoxicity was induced at dose levels >= 800 µg/mL, as shown by a 48 to 100% decrease in the PD.

 

Following the 24-hour treatment, marked to severe cytotoxicity was induced at dose levels >= 600 µg/mL, as shown by a 76 to 100% decrease in the PD.

 

Micronucleus analysis

The dose levels selected for micronucleus analysis were as follows:

.            400, 800 and 1000 µg/mL for the first3-hour treatment, the latter corresponding to the highest tested dose level and inducing a 48% decrease in the PD,

.            400, 600 and 800 µg/mL for the second 3-hour treatment, the latter inducing a 48% decrease in the PD and higher dose levels being too cytotoxic,

.            100, 200 and 400 µg/mL for the 24-hour treatment, the latter inducing a 5% decrease in the PD and higher dose levels being too cytotoxic.

 

Following the 3-hour treatments, the dose levels of 600 µg/mL, 800 µg/mL (second experiment) and 1000 µg/mL (first experiment) showed a frequency of micronucleated cells of both replicate cultures above the corresponding vehicle control historical range (i.e.up to 6‰ vs. [0-3.5] for historical data).

However, since these increases in frequency were neither statistically significant nor dose-related in comparison to the corresponding vehicle controls (p > 0.05), they were considered as not biologically relevant.

 

No statistically significant increase in the frequency of micronucleated cells was noted after the 24-hour treatment. Furthermore, frequencies of micronucleated cells remained consistent with vehicle control reference data and no dose-response relationship was observed (p > 0.05). These results met the criteria of a negative response.

Experiments with S9 mix

The selected dose levels were 200, 400, 600, 1000, 1400, 1750 and 2000 µg/mL.

 

Cytotoxicity

A marked to severe cytotoxicity was induced at dose levels >= 1750 µg/mL, as shown by a 63 to 100% decrease in the PD.

 

Micronucleus analysis

The dose levels selected for micronucleus analysis were 600, 1000 and 1400 µg/mL, the latter inducing an 11% decrease in the PD and higher dose levels being too cytotoxic.

 

No statistically significant increase in the frequency of micronucleated cells was noted after the 3-hour treatment. Moreover, frequencies of micronucleated cells remained consistent with vehicle control historical ranges and no dose-response relationship was observed (p > 0.05).

The recommended level of cytotoxicity was not reached in this experiment, but taking into account the narrow dose levels spacing used, the available results were considered to be suitable to allow a reliable interpretation, and met the criteria of a negative response.

 

Conclusion

The test item did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK^+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

preincubation assay, two different sources of a microsomal metabolic system; 4 instead of 5 tester strains.

Principles of method if other than guideline:

Ames-Test modified acc. to Haworth et al. (1983): Environ. Mutagen. 5, Suppl. 1, 3-142

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Purity = 99%

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Metabolic activation systems were derived from Arochlor-induced livers of male SD rats and male Syrian hamsters.

Test concentrations with justification for top dose:

10, 33, 100, 1000 and 3333 µg/plate
Sec Butylamine was tested in a toxicity assay to determine the appropriate dose-range. Toxic concentrations were those at which a decrease in the number of His+ colonies was seen or at which there was a clearing in the density of the backgroud lawn.

Vehicle / solvent:

Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Test substance well soluble in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: -S9: Sodium azide (TA1535, TA100); 9-aminoacridine (TA1537); 4-nitro-o-phenylenediamine (TA98) // +S9: 2-aminoanthracene (all strains).

Remarks:

4-nitro o phenylendiamine (TA 98)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation);

DURATION
- Preincubation period:20 min
- Exposure duration:2 days at 37 °C
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

NUMBER OF REPLICATIONS:3

The susbtance was tested initially in a toxicity assay to determine the appropriate
dose range. The toxicity assay was pcrformcd by using TA 100 or the system
dcvdoped hy Walch l'i al 119821. Toxic concentrations werc thosc at which a decrcasl'
in the numbcr of his 1 colonies was sccn or at which thcrc was a clearing in the
density of the background lawn.
At least live doses of the chemical wcre tested in triplicate. Experiments were
repcated at least 1 wk following the initial trial. The test susbtance was tested initially at
half-log doses up to a dose that clicited toxicity; subscqucnt trials oo.:asionally uscd
narrowcr dose incrcrncnts. Chcmicals that wcre not toxic were testcd to a maximum
dose of 10 mg/plaie. Chernicals that wcrc poorly soluble wcrc tcsted up to a dose
delïncd by thcir soluhility. A maximum of 0.05 ml sol vent was added to cach plate.
Concurrent solvent and positive controls wcre run with each trial. The positive
controls in the absence of metabolic activation wcrc sodium azide (TA 1535 and
TA 1()()). 9-arninoacridine (IA97 and TA 1537), and 4-nitro-o-phenylcnediamine
(IA98). The positive control for metabolic activation was 2-aminoanthracene for all
strains. Although therc were no specific response ranges established for the solvent
and positive controls. cach laboratory rcjectcd experiments in which the positive
control chcmical did not producc a mutagcnic rcsponse or in which the solvent control
values wcrc highcr (or lowcr in the case of TA 100 and TA97) than their expected
values.
During the initial stage;. of the testing program, chemicals were tested with 10%
S-9. Othcr lcn:ls of S-9 \Vere used when an equivocal resull was ohtained with 10%.
The protocol cvolved to one th!_tl used 30% S-9 when a negative rcsponse was obtained
with I0% S-9.

Evaluation criteria:

Mutagenic (+) or weakly mutagenic (+w) if a reproducible, dose-related increase in revertants over the corresponding solvent controls in replicate trials was seen.
Questionable (?) if a reproducible increase in revertants did not meet the criteria of either "+" or "+w", or if single doses produced an increase in repeat trials.

Species / strain:

other: TA 100, TA 1535, TA 98, TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=100 µg/plate (page 38 table26 of the publication

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

No classification is warranted, all three in vitro tests are negative.