Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 September 2012 to 18 December 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

Detailed under Any other information

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

Detailed under Any other information

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene

EC Number:

272-940-1

EC Name:

Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene

Cas Number:

68921-45-9

Molecular formula:

Variable; substance is a UVCB

IUPAC Name:

Reaction products of N-Phenylbenzenamine with styrene and 2,4,4-trimethylpentene

Test material form:

liquid: viscous

Details on test material:

Identification: Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene
Structure: UVCB
Molecular formula: UVCB
Molecular weight: UVCB
CAS Number: 68921-45-9
Description: Clear red-brown viscous liquid
Batch: EL2B27G336
Purity/Composition: >99%
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 16 February 2014
Stability at higher temperatures: Not indicated
Stability in vehicle: Dimethyl sulfoxide: Unknown
Solubility in vehicle: Dimethyl sulfoxide: Not indicated

Method

Target gene:

chromosome aberrations in cultured peripheral human lymphocytes

Species / strain

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in international guidelines (OECD, EC).

Blood was collected from healthy adult, non-smoking, male volunteers.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and ß-naphthoflavone induced rat liver S9-mix

Test concentrations with justification for top dose:

Without S9-mix :10, 30 and 200 µg/ml culture medium
(24 h exposure time, 24 h fixation time).
10, 30 and 100 µg/ml culture medium
(48 h exposure time, 48 h fixation time).
With S9-mix : 10, 30 and 50 µg/ml culture medium
(3 h exposure time, 48 h fixation time).

Vehicle / solvent:

Dimethyl sulfoxide
Solvent for positive controls: Hanks’ Balanced Salt Solution (HBSS)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

The objective of this study was to evaluate Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene for its ability to induce structural chromosome aberrations in cultured human lymphocytes (1), either in the presence or absence of a metabolic activation system (S9-mix).

Background of the test system: Whole blood samples obtained from healthy male subjects were treated with an anti-coagulant (heparin) and cultured in the presence of a mitogen (phytohaemagglutinin). These stimulated human lymphocytes were used because they are sensitive indicators of clastogenic activity of a broad range of chemicals (1-5).
The stimulated lymphocytes were exposed to the test substance both in the absence and presence of a metabolic activation system (S9-mix). In combination with this metabolic activation system indirect chemical mutagens, i.e. those requiring metabolic transformation into reactive intermediates, can be tested for possible clastogenic effects in vitro.
At predetermined intervals after exposure of the stimulated human lymphocytes to the test substance, cell division was arrested in the metaphase stage of the cell cycle by addition of the metaphase-arresting chemical colchicine. Cells were harvested, stained and metaphase cells were analysed for the presence of structural chromosome aberrations such as breaks, gaps, minutes, dicentrics and exchange figures. Results from cultures treated with the test substance were compared with control (vehicle) treated cultures.
Chromosome aberrations were generally evaluated in the first post-exposure mitosis (i.e. 24 hours after exposure). However, since the appearance of the first post-exposure mitosis could be considerably delayed due to toxic insult to the cells, cells were also harvested 48 hours after exposure to cover the interval in which maximum aberration frequency was expected.
A test substance that induces a positive response in this assay is presumed to be a potential mammalian cell clastogenic agent

Test substance preparation: No correction was made for the purity/composition of the test compound.
Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene was dissolved in dimethyl sulfoxide of spectroscopic quality (SeccoSolv, Merck, Darmstadt, Germany).
Test substance concentrations were used within 3 hours after preparation.
The final concentration of the solvent in the culture medium was 1.0% (v/v).

Reference substances
Negative control: The vehicle for the test substance was dimethyl sulfoxide.

Positive controls
Without metabolic activation (-S9-mix): Mitomycin C (MMC-C; CAS no. 50-07-7, Sigma, Zwijndrecht, The Netherlands) was used as a direct acting mutagen at a final concentration of 0.5 and 0.75 µg/ml for a 3 h exposure period, 0.2 and 0.3 µg/ml for a 24 h exposure period and 0.1 and 0.15 µg/ml for a 48 h exposure period (protocol deviation 1).

With metabolic activation (+S9-mix): Cyclophosphamide (CP; CAS no. 50-18-0. Baxter B.V., Utrecht, The Netherlands) was used as an indirect acting mutagen, requiring metabolic activation, at a final concentration of 10 µg/ml for a 3 h exposure period (24 h fixation time).

Solvent for positive controls: Hanks’ Balanced Salt Solution (HBSS) (Invitrogen Corporation, Breda, The Netherlands), without calcium and magnesium.

All reference stock solutions were stored in aliquots at ≤-15°C in the dark. These solutions were thawed immediately before use.

Test system: Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in international guidelines (OECD, EC).

Blood was collected from healthy adult, non-smoking, male volunteers. The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2011) are presented below:

Dose range finding study: age 41, AGT = 13.9 h (24 h exposure period)
age 35, AGT = 13.9 h (48 h exposure period)
First cytogenetic assay: age 35, AGT = 13.9 h
Second cytogenetic assay: age 33, AGT = 15.8 h (24 and 48 h exposure period)
age 29, AGT = 15.3 h (3 h exposure period)

Cell culture
Blood samples: Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin (Vacuette, Greiner Bio-One, Alphen aan den Rijn, The Netherlands). Immediately after blood collection lymphocyte cultures were started.

Culture medium: Culture medium consisted of RPMI 1640 medium (Invitrogen Corporation), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Invitrogen Corporation), L-glutamine (2 mM) (Invitrogen Corporation), penicillin/streptomycin (50 U/ml and 50 µg/ml respectively) (Invitrogen Corporation) and 30 U/ml heparin (Sigma, Zwijndrecht, The Netherlands).

Lymphocyte cultures: Whole blood (0.4 ml) treated with heparin was added to 5 ml or 4.8 ml culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin (Remel, Europe Ltd., United Kingdom) was added.

Environmental conditions: All incubations were carried out in a controlled environment in the dark, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 58 - 95%), containing 5.0 ± 0.5% CO2 in air, at a temperature of 37.0 ± 1.0°C (actual range 35.0 - 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature (in the range of 35.0 - 36.0°C), humidity (with a maximum of 20%) and CO2 percentage (with a maximum of 1%) occurred that were caused by opening and closing of the incubator door, but the time of these deviations did not exceed 1 hour. Based on laboratory historical data these deviations are considered not to affect the study integrity. Temporary deviation in the humidity in the second cytogenetic assay are explained in protocol deviation 3.

Metabolic activation system
Preparation of S9-fraction: In the dose range finding test and the first and second cytogenetic assay rat liver microsomal enzymes were routinely prepared from adult male Wistar rats (6), which were obtained from Charles River (Sulzfeld, Germany).
The animals were housed at WIL Research Europe in a special room under standard laboratory conditions, as described in the Standard Operating Procedures, and allowed to acclimatise for at least 5 days. The rats were orally dosed at three consecutive days with a suspension of phenobarbital (80 mg/kg body weight; Bufa B.V., IJsselstein, The Netherlands) and ß-naphthoflavone (100 mg/kg body weight; Sigma) in corn oil (they were denied access to food for 3 to 4 hours preceding each dosing). One day after the final exposure (24 h), the rats were sedated using oxygen/carbon dioxide and then killed by decapitation. The rats received a limited quantity of food during the night before sacrifice. The livers of the rats were removed aseptically, and washed in cold (0°C), sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2-EDTA (Merck, Darmstadt, Germany). The livers were minced in a blender and homogenised in 3 volumes of phosphate buffer with a Potter homogeniser. The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-196°C) for a maximum of 1 year.
The S9 batch was characterised in a bacterial reverse mutation assay in Salmonella typhimurium tester strain TA98 with the mutagens Benzo-(a)-pyrene (Sigma) and 2-aminoanthracene (Sigma) at concentrations of 5 µg/plate and 1 µg/plate, respectively. These mutagens require metabolic activation for exerting their mutagenic effects.
In the cytogenetic assay 2A rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg) (see protocol deviation 4).

Preparation of S9-mix: S9-mix was prepared immediately before use and kept on ice. S9-mix components contained per ml: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 µmol HEPES (Invitrogen Corporation).
The above solution was filter (0.22 µm)-sterilized. To 0.5 ml S9-mix components 0.5 ml S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
Metabolic activation was achieved by adding 0.2 ml S9-mix to 5.3 ml of a lymphocyte culture (containing 4.8 ml culture medium, 0.4 ml blood and 0.1 ml (9 mg/ml) phytohaemagglutinin). The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).

Study design
Dose range finding test / First cytogenetic assay: In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test. Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene was tested in the absence and in the presence of 1.8% (v/v) S9-fraction.
Lymphocytes (0.4 ml blood of a healthy male donor was added to 5 ml or 4.8 ml culture medium, without and with metabolic activation respectively and 0.1 ml (9 mg/ml) Phytohaemagglutinin) were cultured for 48 h and thereafter exposed to selected doses of Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene for 3 h, 24 h and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix. A negative control was included at each exposure time.
The highest tested concentration was determined by the solubility of the test substance in the culture medium at the 3 h exposure time. At the 24 and 48 h exposure time, the test substance was tested beyond the limit of solubility to obtain adequate toxicity data.
The test substance precipitated at concentrations of 100 µg/ml and upwards. The lymphocytes were cultured in duplicate at the 3 h exposure time and appropriate vehicle and positive controls were included.
After 3 h exposure to Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium and incubated for another 20 - 22 h (24 h fixation time). The cells that were exposed for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately (24 h and 48 h fixation time).
Cytotoxicity of Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene in the lymphocyte cultures was determined using the mitotic index.
No cytotoxicity was observed in the duplicate cultures of the 3 h exposure time and the slides were scored for chromosome aberrations. The first cytogenetic assay was omitted.
Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the second cytogenetic assay considering the highest dose level had an inhibition of the mitotic index of 50% or greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control.

Second cytogenetic assay: The cytogenetic assay was carried out as described by Evans, 1984 (2) with minor modifications. Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene was tested in the absence and presence of 1.8% (v/v) S9-fraction in duplicate.
Lymphocytes were cultured for 48 h and thereafter exposed in duplicate to selected doses of Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene for 24 h and 48 h in the absence of S9-mix and for 3 h in the presence of S9-mix.
After 3 h exposure, the cells exposed to Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene in the presence of S9-mix were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 ml of HBSS and incubated in 5 ml culture medium for another 44 - 46 h (48 h fixation time).
The cells that were treated for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately after 24 h and 48 h (24 h and 48 h fixation time).
Appropriate negative and positive controls were included in the second cytogenetic assay.

Chromosome preparation: During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 µg/ml medium) (Acros Organics, Geel, Belgium). Thereafter the cell cultures were centrifuged for 5 min at 365 g and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride (Merck) solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol (Merck): acetic acid (Merck) fixative (3:1 v/v).

Preparation of slides: Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the WIL Research Europe study identification number and group number. 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 (Merck) solution in tap water. Thereafter slides were rinsed in tap-water and allowed to dry. The dry slides were automatically embedded in a 1:10 mixture of xylene (Klinipath, Duiven, The Netherlands)/pertex (Klinipath) and mounted with a coverslip in an automated coverslipper (Leica Microsystems B.V., Rijswijk, The Netherlands).

Mitotic index/dose selection for scoring of the cytogenetic assay: The mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells (with a maximum deviation of 5%). At least three analysable concentrations were used for scoring of the cytogenetic assay. Chromosomes of metaphase spreads were analysed from those cultures with an inhibition of the mitotic index of about 50% or above whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations. In case Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene was not cytotoxic and/or difficult to dissolve in aqueous solutions, the highest concentration analysed at the 3 h exposure time was determined by the solubility in the culture medium. If dose related cytotoxicity was observed, the highest concentration analysed at the 24 and 48 h continuous exposure times was based on toxicity irrespective of the solubility of Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene in the culture medium. However, the extent of precipitation may not interfere with the scoring of chromosome aberrations.

Analysis of slides for chromosome aberrations: To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with WIL Research Europe study identification number and code was placed over the marked slide. One hundred metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 25 in 50 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with aberrations and the number of aberrations were calculated.

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics:

(N-1) (ad-bc)2
X2 = --------------------------------
(a+b) (c+d) (a+c) (b+d)

where b = the total number of aberrant cells in the control cultures.
d = the total number of non aberrant cells in the control cultures.
n0 = the total number of cells scored in the control cultures.
a = the total number of aberrant cells in treated cultures to be compared with the control.
c = the total number of non aberrant cells in treated cultures to be compared with the control.
n1 = the total number of cells scored in the treated cultures.
N = sum of n0 and n1
(N-1) (ad-bc)2
If P X2 > ------------------------------- (one-tailed) is small (p< 0.05) the hypothesis that the
(a+b) (c+d) (a+c) (b+d)

incidence of cells with chromosome aberrations is the same for both the treated and the solvent control group is rejected and the number of aberrant cells in the test group is considered to be significantly different from the control group at the 95% confidence level.

Results and discussion

Test results

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Dose range finding test / First cytogenetic assay: At a concentration of 100 µg/ml the test substance precipitated in the culture medium. In the dose range finding study, at the 3 h exposure time, blood cultures were treated in duplicate with 10, 33 and 100 µg test substance/ml culture medium with and without S9-mix (first cytogenetic assay).
At the 24 h and 48 h continuous exposure time blood cultures were treated with 1, 3, 10, 33, 100, 333 and 1000 µg test substance/ml culture medium without S9-mix. The test substance was tested beyond the limit of solubility to obtain adequate toxicity data.
All dose levels were selected for scoring of chromosome aberrations.
Both in the absence and presence of S9-mix, Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.
Both in the absence and presence of S9-mix, Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

Second cytogenetic assay: To obtain more information about the possible clastogenicity of Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to the test substance in the absence of S9-mix for 24 or 48 hours. In the presence of S9-mix, cells were fixed after 48 hours following a 3 hour exposure to Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene. Further investigation showed that a concentration of 50 µg/ml already precipitated in the culture medium. The following dose levels were selected for the second cytogenetic assay:

Without S9-mix: 1, 3, 10, 30, 50, 100, 200 and 300 µg/ml culture medium
(24 and 48 h exposure time, 24 and 48 h fixation time).
With S9-mix: 10, 30 and 50 µg/ml culture medium
(3 h exposure time, 48 h fixation time).

Based on these observations the following doses were selected for scoring of chromosome aberrations:

Without S9-mix: 10, 30 and 200 µg/ml culture medium
(24 h exposure time, 24 h fixation time).
10, 30 and 100 µg/ml culture medium
(48 h exposure time, 48 h fixation time).
With S9-mix: 10, 30 and 50 µg/ml culture medium
(3 h exposure time, 48 h fixation time).

Both in the absence and presence of S9-mix, Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.
Both in the absence and presence of S9-mix, Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

Chemical analysis of dose preparations: The concentrations analysed in the samples prepared for use were in agreement with the nominal concentrations (i.e. mean accuracies 88 and 93%). Analysis of low and high formulations after storage yielded a relative difference of ≤ 10%. Based on this, the formulations were found to be stable when stored at room temperature under normal laboratory light conditions for at least 4 hours.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Chromosome aberrations in human lymphocyte cultures treated with Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene

 

In the absence of S9-mix in the second cytogentic assay (24h exposure time, 24 h fixation time)

Conc	DMSO (1.0% v/v)			10  μg/ml			30 μg/ml			200  μg/ml			MMC-C 0.2μg/ml
Culture	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B
Mitotic Index (%)	100			92			87			66			47
No. of Cells scored	100	100	200	100	100	200	100	100	200	100	100	200	100	100	200
No. of Cells with aberrations (+ gaps)^a)	1	0	1	0	1	1	0	0	0	1	0	1	32	29	^***)61
No. of Cells with aberrations (- gaps)	1	0	1	0	1	1	0	0	0	1	0	1	30	25	^***)55
g’													2	3
g”													4	3
b’										1			14	7
b”					1								15	10
m’	1												4	2
m”													1	3
exch.													4	3
dic
d’
misc.
total aberr (+ gaps)	1	0		0	1		0	0		1	0		44	31
total aberr  (- gaps)	1	0		0	1		0	0		1	0		38	25

 

In the absence of S9-mix in the second cytogenetic assay (48h exposure time, 48h fixation time)

Conc	DMSO (1.0% v/v)			10 μg/ml			30 μg/ml			100 μg/ml			MMC-C  0.1μg/ml
Culture	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B
Mitotic Index (%)	100			93			89			32			53
No. of Cells scored	100	100	200	100	100	200	100	100	200	100	100	200	100	100	200
No. of Cells with aberrations (+ gaps)^a)	0	0	0	0	0	0	0	0	0	1	0	1	28	28	***) 56
No. of Cells with aberrations (- gaps)	0	0	0	0	0	0	0	0	0	1	0	1	24	24	***) 48
g’													3	3
g”													2	4
b’										1			5	6
b”													10	11
m’													1	2
m”													4	3
exch.													5	1
dic
d’														1
misc.				poly	2poly					poly			p	p,poly
total aberr (+ gaps)	0	0		0	0		0	0		1	0		31	32
total aberr (- gaps)	0	0		0	0		0	0		1	0		26	25

The numerical variation polyploidy (poly) was not counted as an aberration.

 

In the presence of S9-mix in the second cytogenetic assay (3h exposure time, 48h fixation time)

Conc	DMSO (1.0% v/v)			10 μg/ml			30 μg/ml			50 μg/ml			CP 10 μg/ml
Culture	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B
Mitotic Index (%)	100			109			129			116			-^b)
No. of Cells scored	100	100	200	100	100	200	100	100	200	100	100	200	100	100	200
No. of Cells with aberrations (+ gaps)^a)	0	0	0	1	0	1	1	0	1	0	0	0	30	29	***) 59
No. of Cells with aberrations (- gaps)	0	0	0	1	0	1	0	0	0	0	0	0	28	28	***) 56
g’							1							1
g”													2	1
b’				1									16	14
b”													17	11
m’														1
m”														1
exch.													2	4
dic
d’
misc.
total aberr (+ gaps)	0	0		1	0		1	0		0	0		37	33
total aberr (- gaps)	0	0		1	0		0	0		0	0		35	31

^a)Abbreviations use for various types of aberrations are listed in APPENDIX 2 (detailed under Overall Remarks). Misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.

^b)CP was fixed after 24 hours. Therefore, the mitotic index could not be calculated as percentage of control.

*) Significantly different from control group (Chi-square test), *P < 0.05, **P < 0.01 or ***P < 0.001.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

It is concluded that this test is valid and that Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene is not clastogenic in human lymphocytes under the experimental conditions described in the report.

Executive summary:

Evaluation of the ability of Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene to induce chromosome aberrations in cultured peripheral human lymphocytes (with repeat experiment).

 

This report describes the effect of Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity of the test substance was tested in two independent experiments.

 

The study procedures described in this report were based on the most recent OECD and EC guidelines.

 

Batch EL2B27G336 of Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene was a clear red-brown viscous liquid. The test substance was dissolved in dimethyl sulfoxide.

 

The concentrations analysed in the samples prepared for use were in agreement with the nominal concentrations (i.e. mean accuracies 88 and 93%). Analysis of low and high formulations after storage yielded a relative difference of ≤ 10%. Based on this, the formulations were found to be stable when stored at room temperature under normal laboratory light conditions for at least 4 hours.

 

In the first cytogenetic assay, Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene was tested up to 100 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. The test substance precipitated in the culture medium at this dose level.

 

In the second cytogenetic assay, the test substance was tested up to 200 µg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 100 µg/ml for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels. In the presence of S9-the test substance was tested up to 50 µg/ml for a 3 h exposure time with a 48 h fixation time. The test substance precipitated in the culture medium at this dose level.

 

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

 

Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene did not induce a 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 repeated experiments.

 

No effects of the test substance 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 Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

 

Finally, it is concluded that the test is valid and that Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4 -trimethylpentene is not clastogenic in human lymphocytes under the experimental conditions described in the report.