4,4'-methylenebis[N-sec-butylaniline]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A chromosome aberration study with 4,4’-methylenebis-N-sec-butylaniline was performed according to OECD guideline 473 and GLP principles. A bacterial reverse mutation test was done according to OECD guideline 471 and GLP principles. Furthermore, an in vitro mammalian cell gene mutation test was performed according to OECD guideline 490 and GLP principles with 4,4'-methylenebis-N-sec-butylaniline. As 4,4'-methylenebis-N-sec-butylaniline gave a negative result in all three tests, there are no indications that it has genotoxic properties.

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:

06 August 2018 - 17 September 2018

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:

29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Source of cells: L5178Y/TK+/- -3.7.2C mouse lymphoma cells from American Type Culture Collection, (ATCC, Manassas, USA), (2001)
- Suitability of cells: recommended test system in international guidelines

MEDIA USED
- Type and identity of media
Horse serum: inactivated by incubation at 56°C for at least 30 minutes
Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin
Growth medium: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Exposure medium: 3 hr exposure: basic medium, supplemented with 5% (v/v) heat-inactivated horse serum (=R5 medium); 24 hr exposure: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 μg/mL trifluorothymidine (TFT)
Non-selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium)
- Properly maintained: yes, stock cultures of the cells were stored in liquid nitrogen (-196°C).
- Periodically checked for Mycoplasma contamination: yes
- Cleansed against high spontaneous background: yes

ENVIROMENTAL CONDITIONS
- All incubations were carried out in a humid atmosphere All incubations were carried out in a humid atmosphere (80 - 100%, actual range 52 -99%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.1 - 37.5°C).

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).

Test concentrations with justification for top dose:

Dose-range finding test (with and without S9-mix, 3 hour treatment; without S9-mix, 24 hour treatment): 9.8, 20, 39, 78, 156, and 313 μg/mL

Main experiment 1
3 hour treatment (Without S9-mix): 0.31, 0.63, 1.25, 2.5, 5, 10, 20, 30, 35 and 40 μg/mL exposure medium.
3 hour treatment (With S9-mix): 2.5, 5, 10, 20, 40, 60, 80, 90, 100, 120 and 140 μg/mL exposure medium.

Main experiment 1a
3 hour treatment (without S9-mix): 0.63, 1.25, 2.5, 5, 10, 20, 30, 35, 40, 50, 60, 70 and 80 μg/mL exposure medium

Main experiment 2
24 hour treatment (without S9): 1.25, 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 μg/mL exposure medium.

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: Based on a solubility test, DMSO was selected as vehicle.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9; in DMSO, 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9; 7.5 µg/mL in Hanks’ balanced salt solution without calcium and magnesium.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: below 1 x 10^6 cells/mL

TEST ITEM PREPARATION
The test item was dissolved in DMSO. Test item concentrations were used within 1 hour after preparation.


DURATION
- Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10^-4 M hypoxanthine, 2 x 10^-7 M aminopterine and 1.6 x 10^-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10-medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment.
- Exposure duration: 3 hours (experiment 1; with and without S9-mix); 24 hours (experiment 2; without S9-mix).
- Expression time: 2 days in which at least 4 x 10^6 cells were subcultured every day
- Selection time (if incubation with a selection agent): 11 or 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)
STAIN: 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)

NUMBER OF REPLICATIONS:
- test concentrations: 1
- positive control: 1
- solvent control: 2

DETERMINATION OF THE MUTATION FREQUENCY
For determination of the cloning efficiency the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
For determination of the mutation frequency (MF) a total number of 9.6 x 10^5 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups where a total number of 9.6 x 10^5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium.
The microtiter plates for cloning efficiency and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 1.5-2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to each well. The plates for the cloning efficiency and MF were scored with the naked eye or with the microscope.

ACCEPTABILITY CRITERIA
A mutation assay was considered acceptable if it met the following criteria:

a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10^-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10^-6).

Evaluation criteria:

EVALUATION CRITERIA:
a) Any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
b) The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

DOSE-RANGE FINDING STUDY

Precipitation observed:
78 μg/mL and above directly after adding.
156 μg/mL and above after 3 hours treatment.
313 μg/mL after 24 hours treatment.

3 hours treatment without S9-mix
Relative suspension growth was 14% at a dose level of 39 μg/mL compared to the relative suspension growth of the solvent control.
No significant cell survival was observed at test item concentrations of 78 μg/mL and above.

3 hours treament with S9-mix
Relative suspension growth was 29% at a dose level of 78 μg/mL compared to the relative suspension growth of the solvent control.
No significant cell survival was observed at test item concentrations of 156 μg/mL and above.

24 hours treatment without S9-mix
The relative suspension growth was 5% at the test item concentration of 39 μg/mL compared to the relative suspension growth of the solvent control.
No significant cell survival was observed at test item concentrations of 78 μg/mL and above.

MAIN EXPERIMENT 1

In the 3 hours treatment without S9-mix, no dose level with a cell survival below 46% was reached, therefore this experiment was rejected and replaced by experiment 1a.

The dose levels of 0.63 to 20 μg/mL showed no cytotoxicity. Therefore, the dose levels of 2.5 and 20 μg/mL were not regarded relevant for mutation frequency measurement. The dose levels of 35 and 40 μg/mL showed similar cell growth delay. Therefore, the dose level of 40 μg/mL was not regarded relevant for mutation frequency measurement. The dose levels of 70 and 80 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.

In the presence of S9-mix, the dose levels of 2.5 to 40 μg/mL showed no cytotoxicity. Therefore, the dose levels of 2.5 and 10 μg/mL were not regarded relevant for mutation frequency measurement. The dose levels of 120 and 140 μg/mL showed similar cell growth delay. Therefore, the dose level of 120 μg/mL was not regarded relevant for mutation frequency measurement.

The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 0.63, 1.25, 5, 10, 30, 35, 50 and 60 μg/mL exposure medium.
With S9-mix: 5, 20, 40, 60, 80, 90, 100 and 140 μg/mL exposure medium.

Precipitation observed:
Without S9-mix: no precipitation
With S9-mix: 140 μg/mL.

The relative total growth of the highest test item concentration was 12% compared to the total growth of the solvent controls both in the absence and presence of S9-mix.

No biologically relevant increase in the mutation frequency at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.


MAIN EXPERIMENT 2

The dose levels of 25 and 30 μg/mL showed similar cell growth delay. Therefore, the dose level of 25 μg/mL was not regarded relevant for mutation frequency measurement. The dose levels of 40 to 50 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.

The dose levels selected to measure mutation frequencies at the TK-locus were:
1.25, 2.5, 5, 10, 15, 20, 30 and 35 μg/mL exposure medium.

The relative total growth of the highest test item was 15% compared to the total growth of the solvent control.

No biologically relevant increase in the mutation frequency at the TK locus was observed after treatment with the test item. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

CONTROLS

The mutation frequency of one of the solvent control cultures in two experiments were recorded to be outside the range of ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors (41 and 184 per 10^6 survivors in the first and second main experiment, respectively). The duplicate solvent controls were within the acceptability criteria (68 and 165 per 10^6 survivors in the first and second main experiment, respectively). According to the guideline a single solvent control group is sufficient. Hence determination of the mutagenicity of the test item with only one solvent control had no effect on the reliability of the results in the experiment.
The suspension growth over the two-day expression period for cultures treated with DMSO was between 15 and 23 (3 hour treatment) and 80 and 85 (24 hour treatment).
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Conclusions:

An in vitro mammalian cell gene mutation test was performed according to OECD guideline 490 and GLP principles with 4,4'-methylenebis-N-sec-butylaniline. Based on the results, 4,4'-methylenebis-N-sec-butylaniline is not mutagenic with and without metabolic activation in the TK mutation test system under the experimental conditions described in this report.

Executive summary:

An in vitro Mammalian Cell Gene Mutation Test using the Thymidine Kinase Gene was done according to OECD guideline 490 and GLP principles in order to evaluate the mutagenic potential of 4,4’-methylenebis-N-sec-butylaniline. The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay. In addition, positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation demonstrating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. In the absence of S9-mix, 4,4’-methylenebis-N-sec-butylaniline did not induce a biologically relevant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, 4,4’-methylenebis-N-sec-butylaniline did not induce a biologically relevant increase in the mutation frequency. In conclusion, 4,4’-methylenebis-N-sec-butylaniline is not mutagenic in the mouse lymphoma L5178Y test system.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 March 2018 - 11 April 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix induced by Aroclor 1254 (500 mg/kg bw)

Test concentrations with justification for top dose:

Direct plate assay
Dose-range finding test (without and with S9; tester strains TA100 and WP2uvrA): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate (reported as part of experiment 1)
First experiment (without and with S9; tester strains TA1535, TA1537 and TA98): 5.4, 17, 52, 164, 512, 1600 μg/plate

Pre-incubation assay
Second experiment (without and with S9, tester strains WP2uvrA and TA100): 1.7, 5.4, 17, 52, 164, 512, 1600 μg/plate
Second experiment (without and with S9, tester strains TA1535, TA1537, TA 98): 1.7, 5.4 17, 52, 164, 512, μg/plate

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: a previously performed solubility test showed that the test item was dissolved in DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Remarks:

For details on positive control substances, see Table 1 and Table 2

Details on test system and experimental conditions:

Two individual experiments were performed. The dose range-finding study with tester strains TA100 and WP2uvrA was reported as part of the first experiment. The first experiment was a direct plate assay. The second experiment was a pre-incubation assay and was performed to obtain more information about the possible mutagenicity of the test item.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period (second experiment): 30 ± 2 minutes at 70 rpm at 37 ± 1°C
- Exposure duration: 48 ± 4 h (in the dark at 37.0 ± 1.0 °C)

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain.

METHODS: The following solutions were successively added to 3 mL molten top agar: 0.1 ml of a dilution of the test item in DMSO and either 0.5 ml S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. In the pre-incubation assay (second experiment) this procedure was preceeded by pre-incubation of the solutions.

DETERMINATION OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.
- Other: precipitation of the test item was recorded

COLONY COUNTING
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

ACCEPTABILITY CRITERIA
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the direct method, with and without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the pre incubation method, without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRECIPITATION
- Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 1600 µg/plate and upwards and at 512 µg/plate and above at the end of the incubation period.
- Precipitation of the test item on the plates was observed at the start of the incubation period at the concentration of 512 µg/plate in tester strains TA1535, TA1537 and TA98 in the absence of S9-mix. Precipitate was observed at the end of the incubation period at the top dose levels tested in all five tester strains, except in tester strain TA100 in the presence of S9-mix where no precipitate was observed.

- There were no increases in the number of revertant colonies indicating a positive repsonse for inducing mutagenicity.
- The negative and strain-specific positive control values were within the laboratory historical control data ranges
- In both the first and second assay, criteria for a negative response were met for all tester strains with and without metabolic activation.

Conclusions:

Based on the results of an Ames test, performed according to OECD guideline 471 and GLP principles, 4,4’-methylenebis-N-sec-butylaniline is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

A bacterial reverse mutation test was done according to OECD guideline 471 and GLP principles. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test item did not induce a significant dose-related increase in the number of revertant (His⁺) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp⁺) colonies in tester strain WP₂uvrA both in the absence and presence of S9-metabolic activation. The selected dose-range for the test item included cytotoxic and precipitating concentrations. These results were confirmed in a follow-up experiment. In conclusion, based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia colireverse mutation assay. 

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 March 2018 - 30 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Peripheral human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED:
- Source of cells: blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
-Suitability of cells: Cells were collected from healthy adult, non-smoking volunteers (approximately 18 to 35 years of age).
- Average Generation Time (AGT) : the cells and the age of the donor at the time the AGT was determined are presented below:

Dose-range finding study: age 32, AGT = 14.6 h
First cytogenetic assay: age 24, AGT = 13.0 h
Second cytogenetic assay: age 30, AGT = 14.0 h

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 medium supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) and 30 U/mL heparin

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw).

Test concentrations with justification for top dose:

The highest concentration analyzed was selected based on the solubility of the test item in the culture medium.

- Dose range finding study
3 h exposure, 24 h fixation, with and without S9: 7.8, 15.6, 31.3, 62.5, 125, 250 μg/mL
24 h exposure, 24 h fixation, without S9: 15.6, 31.3, 62.5, 125, 250, 500 μg/mL
48 h exposure, 48 h fixation, without S9: 15.6, 31.3, 62.5, 125, 250, 500 μg/mL

- First cytogenetic assay:
3 h exposure, 24 h fixation, with and without S9: 5, 50, 100, 150, 200, 250 μg/mL

- Second cytogenetic assay:
24 h exposure, 24 h fixation, without S9: 1, 10, 20, 40, 60, 80, 100 μg/mL
48 h exposure, 48 h fixation, without S9: 1, 10, 20, 40, 60, 80, 100 μg/mL

Test item concentrations were used within 3 hours after preparation.

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent: The test item was dissolved in DMSO, which is recommended by international guidelines as solvent.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9

Details on test system and experimental conditions:

Two independent cytogenetic assays were performed, preceeded by a dose-range finding assay.

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration experiment 1: 3 h (with and without S9)
- Exposure duration experiment 2: 24, 48 h (without S9)
- Fixation time: 24 h (for 3 or 24 hour exposure period) and 48 h (for 48 hour exposure period)

RINSING AFTER EXPOSURE
3 hour exposure: yes
24 hour exposure: no
48 hour exposure: no

ENVIRONMENTAL CONDITIONS:
- Humidity: 44 - 89%
- Temperature: 34.9 - 37.1 °C

SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.5 μg/ml medium)

STAIN (for cytogenetic assays): Giemsa (5% v/v)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated coverslipper.

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 1000

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: Between 48 and 150 in each replicate

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (the highest concentration analyzed was determined by the solubility in the culture medium)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreduplication: yes

Evaluation criteria:

ACCEPTANCE CRITERIA
A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the number of cells with chromosome aberrations. The positive control data will be analyzed by the Fisher’s exact test (one-sided, p < 0.05).


EVALUATION CRITERIA
A test item is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, onesided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Key result

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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: No
- Effects of osmolality: No

DOSE LEVELS SELECTED FOR SCORING:
Dose levels were selected based on toxicity, inhibition of the mitotic index of about 50% or greater.
- First cytogenetic assay: 5, 100 and 200 μg/mL were selected for scoring.
- Second cytogenetic assay: 1, 10 and 40 μg/mL were selected for scoring.

RANGE-FINDING/SCREENING STUDIES:
- The test item precipitated in culture medium with 3 hour exposure, with and without S9 at a concentration of 250 μg/mL
- The test item precipitated in culture medium with 24 and 48 hour exposure at a concentration of 500 μg/mL.

FIRST CYTOGENETIC ASSAY
- Precipitation in the test: In the first cytogenetic test, the test item precipitated in the culture medium at 200 µg/mL and upwards.
- Both in the absence and presence of S9-mix, the test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.
- The test item did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

SECOND CYTOGENETIC ASSAY
- The test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.
- The test item did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.


COMPARISON WITH HISTORICAL CONTROL DATA AND VALIDITY:
- The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals both produced statistically significant increases in the frequency of aberrant cells. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the
distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Conclusions:

A chromosome aberration study with 4,4’-methylenebis-N-sec-butylaniline was performed according to OECD guideline 473 and GLP principles. Based on the results of two independent experiments in cultured peripheral human lymphocytes, it was concluded that 4,4’-methylenebis-N-sec-butylaniline is not clastogenic in human lymphocytes under these experimental conditions.

Executive summary:

An in vitro mammalian chromosome aberration test was done according to OECD guideline 473 and GLP principles. The objective of this study was to evaluate 4,4’-methylenebis-N-sec-butylaniline for its ability to induce structural chromosome aberrations in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix). The possible clastogenicity of the test item was tested in two independent experiments. The vehicle of the test item was dimethyl sulfoxide. In the first cytogenetic assay the test item was tested up to 200 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. Based on the results of appropriate controls, it was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. The highest tested concentration was determined by the solubility of the test item in the culture medium. The test item did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments. No biologically relevant effects of the test item on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore, it can be concluded that the test item does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

An in vitro mammalian chromosome aberration test was done according to OECD guideline 473 and GLP principles. The objective of this study was to evaluate 4,4’-methylenebis-N-sec-butylaniline for its ability to induce structural chromosome aberrations in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix). The possible clastogenicity of the test item was tested in two independent experiments. The vehicle of the test item was dimethyl sulfoxide. In the first cytogenetic assay the test item was tested up to 200 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. Based on the results of appropriate controls, it was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. The highest tested concentration was determined by the solubility of the test item in the culture medium. The test item did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments. No biologically relevant effects of the test item on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore, it can be concluded that the test item does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations.

A bacterial reverse mutation test was done according to OECD guideline 471 and GLP principles. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test item did not induce a significant dose-related increase in the number of revertant (His⁺) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp⁺) colonies in tester strain WP₂uvrA both in the absence and presence of S9-metabolic activation. The selected dose-range for the test item included clearly toxic and precipitating concentrations. These results were confirmed in a follow-up experiment. In conclusion, based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia colireverse mutation assay. 

An in vitro Mammalian Cell Gene Mutation Test using the Thymidine Kinase Gene was done according to OECD guideline 490 and GLP principles in order to evaluate the mutagenic potential of 4,4’-methylenebis-N-sec-butylaniline. The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay. In addition, positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation demonstrating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. In the absence of S9-mix, 4,4’-methylenebis-N-sec-butylaniline did not induce a biologically relevant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, 4,4’-methylenebis-N-sec-butylaniline did not induce a biologically relevant increase in the mutation frequency. In conclusion, 4,4’-methylenebis-N-sec-butylaniline is not mutagenic in the mouse lymphoma L5178Y test system.