Formaldehyde, oligomeric reaction products with acetone and diphenylamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 April 2016 to 14 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Guideline no. 473: In Vitro Mammalian Chromosome Aberration Test (adopted September 26, 2014).

Deviations:

no

GLP compliance:

yes

Type of assay:

other: chromosome aberrations

Test material

Test material information

Constituent 1

Reference substance name:

Formaldehyde, oligomeric reaction products with acetone and diphenylamine

EC Number:

500-011-5

EC Name:

Formaldehyde, oligomeric reaction products with acetone and diphenylamine

Cas Number:

9003-80-9

Molecular formula:

UVCB substance - not applicable

IUPAC Name:

N-phenylaniline; formaldehyde; propan-2-one

Test material form:

solid: flakes

Details on test material:

6.1. Test item
6.1.1. Test item information
Identification Formaldehyde, oligomeric reaction products with acetone
and diphenylamine
Appearance Dark brown flakes
Batch IC5B04P006
Purity/Composition 100% Unknown or Variable compositions, Complex reaction products and Biological materials (UVCB)
Test item storage At room temperature
Stable under storage conditions until 26 February 2019 (expiry date)

Specific details on test material used for the study:

Test item: 206534/AIdentification: Formaldehyde, oligomeric reaction products with acetone and diphenylamineAppearance: Dark brown flakesBatch: IC5B04P006Purity/Composition: 100% Unknown or Variable compositions, Complex reaction products and Biological materials (UVCB)Test substance storage: At room temperatureStable under storage conditions until: 26 February 2019 (expiry date)Purity/composition correction factor: No correction factor requiredTest substance handling: No specific handling conditions requiredStability at higher temperatures: Not availableChemical name (IUPAC), synonym or trade name: Formaldehyde, oligomeric reaction products with acetone and diphenylamine (BXA)CAS Number: 9003-80-9Molecular structure: UVCBMolecular formula: UVCBMolecular weight: UVCBpH (1% in water, indicative range): 7 .76 – 7.37 (determined by Charles River Den Bosch )

Method

Target gene:

structural chromosome aberrations

Species / strain

Species / strain / cell type:

lymphocytes: Cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

Blood was collected from healthy adult, non-smoking volunteers (approximately 18 to 35 years of age). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2015) are presented below:Dose range finding study: age 35, AGT = 12.9 h (24 hours exposure time)Dose range finding study: age 28, AGT = 13.4 h (48 hours exposure time)First cytogenetic assay: age 35, AGT = 12.9 hSecond cytogenetic assay: age 33, AGT = 12.7 hCell cultureBlood samples: Blood samples were collected by venepuncture 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 (Life technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Life technologies), L-glutamine (2 mM) (Life technologies), penicillin/streptomycin (50 U/ml and 50 μg/ml respectively) (Life technologies) 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., Dartford, United Kingdom) was added.Environmental conditions: All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 55 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.9 - 37.2°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, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Dose range finding test / First cytogenetic assay3 h exposure time, 17, 52 and 164 μg test item/ml (with and without S9-mix)24 h and 48 h exposure time, 1.7, 5.4, 17, 52 and 164 μg test item/ml (without S9-mix)Second cytogenetic assayWithout S9-mix : 17, 52 and 164 μg/ml culture medium (24 and 48 h exposure time, 24 and 48 h fixation time).In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test. Formaldehyde, oligomeric reaction products with acetone and diphenylamine was tested in the absence and in the presence of 1.8% (v/v) S9-fraction.

Vehicle / solvent:

dimethyl sulfoxide

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:

Metabolic activation systemRat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is 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). Preparation of S9-mixS9-mix was prepared immediately before use and kept on ice. S9-mix components contained per ml physiological saline: 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 (Life technologies).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 designDose range finding test / First cytogenetic assayIn order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test. Formaldehyde, oligomeric reaction products with acetone and diphenylamine was tested in the absence and in the presence of 1.8% (v/v) S9-fraction.Lymphocytes (0.4 ml blood of a healthy 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 Formaldehyde, oligomeric reaction products with acetone and diphenylamine 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 Formaldehyde, oligomeric reaction products with acetone and diphenylamine in the culture medium.The test item precipitated at concentrations of 164 μg/ml and upwards. The lymphocytes were cultured in duplicate at the 3 h exposure time and appropriate positive controls were included. The cytogenetic assay was carried out as described by Evans, 1984 (2) with minor modifications. Formaldehyde, oligomeric reaction products with acetone and diphenylamine was tested in the absence and presence of 1.8% (v/v) S9-fraction in duplicate.After 3 h exposure to Formaldehyde, oligomeric reaction products with acetone and diphenylamine 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 re-suspended 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 Formaldehyde, oligomeric reaction products with acetone and diphenylamine 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 pilot study (short term exposure period) was used as the first cytogenetic assay.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 was determined by the solubility. As clear negative results were obtained in the presence of metabolic activation, the repetition of the experiment was not considered necessary.Second cytogenetic assayTo confirm the results of the first cytogenetic assay a second cytogenetic assay was performed with an extended exposure time of the cells in the absence of S9-mix.Lymphocytes were cultured for 48 ± 2 h and thereafter exposed in duplicate to selected doses of Formaldehyde, oligomeric reaction products with acetone and diphenylamine for 24 h and 48 h in the absence of S9-mix.The cells 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 preparationDuring 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 slidesFixed 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 Charles River Den Bosch 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 Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded in a 1:10 mixture of xylene (Klinipath, Duiven, The Netherlands)/pertex (Histolab, Gothenburg, Sweden) and mounted with a coverslip in an automated cover slipper (Leica Microsystems B.V., Rijswijk, The Netherlands).Mitotic index/dose selection for scoring of the cytogenetic assayThe 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. The test item was not severe cytotoxic, the highest concentration analysed was determined by the solubility in the culture medium.Analysis of slides for chromosome aberrationsTo prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with Charles River Den Bosch study identification number and code was placed over the marked slide. One hundred and fifty metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 38 in 75 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. Since the lowest concentration of MMC-C resulted in a positive response the highest concentration was not examined for chromosome aberrations.

Rationale for test conditions:

Background of the test systemWhole blood samples obtained from healthy 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.The stimulated lymphocytes were exposed to Formaldehyde, oligomeric reaction products with acetone and diphenylamine 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 Formaldehyde, oligomeric reaction products with acetone and diphenylamine, 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 Formaldehyde, oligomeric reaction products with acetone and diphenylamine were compared with control (vehicle) treated cultures.Chromosome aberrations are 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 item that induces a positive response in this assay is presumed to be a potential mammalian cell clastogenic agent.

Evaluation 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 analysed by the Fisher’s exact test (one-sided, p < 0.05).

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.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, one sided, 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.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: Cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

164 μg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Dose range finding test / First cytogenetic assayAt a concentration of 164 μg/ml Formaldehyde, oligomeric reaction products with acetone and diphenylamine precipitated in the culture medium. At the 3 h exposure time, blood cultures were treated in duplicate with 17, 52 and 164 μg test item/ml culture medium with and without S9-mix (first cytogenetic assay).At the 24 hour and 48 hour exposure time single blood cultures were treated with 1.7, 5.4, 17, 52 and 164 μg Formaldehyde, oligomeric reaction products with acetone and diphenylamine/ml culture medium without S9-mix (dose range finding test).Both in the absence and presence of S9-mix, Formaldehyde, oligomeric reaction products with acetone and diphenylamine did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. MLA-3202 did not induce a biologically relevant increase the number of polyploid cells and cells with endoreduplicated chromosomes. Although 1 endoreduplicated chromosome was observed at the lowest concentration and 5 polyploid cells in the highest concentration in the presence of S9-mix, which both are outside the 95% control limits of the distribution of the historical negative control database, these were only observed in one single culture and no dose relationship was observed. Therefore these are considered isolated events and therefore considered not biologically relevant.Second cytogenetic assayTo obtain more information about the possible clastogenicity of Formaldehyde, oligomeric reaction products with acetone and diphenylamine, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to Formaldehyde, oligomeric reaction products with acetone and diphenylamine in the absence of S9-mix for 24 or 48 hours. The following dose levels were selected for the second cytogenetic assay:Without S9-mix : 17, 52 and 164 μg/ml culture medium (24 and 48 h exposure time, 24 and 48 h fixation time).Based on these observations all dose levels were selected for scoring of chromosome aberrations.Formaldehyde, oligomeric reaction products with acetone and diphenylamine did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.Formaldehyde, oligomeric reaction products with acetone and diphenylamine did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

Any other information on results incl. tables

Mitotic index of human lymphocyte cultures treated with Formaldehyde, oligomeric

reaction products with acetone and diphenylamine in the dose range finding study

Test item concentration (μg/ml)	Number of metaphases
	Absolute	Number of cells scored	Percentage of control
24 h exposure time, 24 h fixation time
Control^a) 1.7 5.4 17 52 164^b)	40 33 37 31 38 33	1000 1000 1000 1000 1002 1000	100 83 93 78 95 83
48 h exposure time, 48 h fixation time
Control^a) 1.7 5.4 17 52 164^b)	80 77 79 85 82 66	1000 1004 1005 1000 1004 1004	100 96 99 106 103 83

a) Dimethyl sulfoxide

b) Formaldehyde, oligomeric reaction products with acetone and diphenylamine precipitated in the culture medium

 

Mitotic index of human lymphocyte cultures treated with Formaldehyde, oligomeric

reaction products with acetone and diphenylamine in the first cytogenetic assay

Test item concentration (μg/ml)	Number of metaphases^a)
	Absolute	Number of cells scored	Percentage of control
Without metabolic activation (-S9-mix)
3 h exposure time, 24 h fixation time
Control^b) 17 52 164^c) MMC-C; 0.5μg/ml MMC-C; 0.75μg/ml	54 – 64 64 – 64 54 – 72 51 – 51 37 – 39 25 – 34	1000 – 1000 1000 – 1000 1000 – 1000 1000 – 1000 1000 – 1000 1000 – 1000	100 108 107 86 64 50
With metabolic activation (+S9-mix)
3 h exposure time, 24 h fixation time
Control^b) 17 52 164^c) CP; 10μg/ml	80 – 74 58 – 78 65 – 63 64 – 56 31 – 36	1000 – 1001 1000 – 1000 1000 – 1000 1000 – 1000 1000 – 1000	100 88 83 78 44

a) Duplicate cultures

b) Dimethyl sulfoxide

c) Formaldehyde, oligomeric reaction products with acetone and diphenylamine precipitated in the culture medium

 

Chromosome aberrations in human lymphocyte cultures treated with Formaldehyde, oligomeric reaction products with

acetone and diphenylamine in the absence of S9-mix in the first cytogenetic assay (3 h exposure time, 24 h fixation time)

Conc	DMSO (1.0% v/v)			17μg/ml			52μg/ml			164μg/ml			MMC-C 0.5μg/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			108			107			86			64
No. of Cells scored	150	150	300	150	150	300	150	150	300	150	150	300	150	150	300
No. of Cells with aberrations (+ gaps)^a)	1	1	2	0	0	0	2	0	2	2	0	2	32	25	^***)57
No. of Cells with aberrations (- gaps)	1	1	2	0	0	0	2	0	2	2	0	2	32	24	^***)56
g'														2
g'’
b'	1						2			2			25	16
b'’		1
m'
m'’
exch.													9	9
dic.
d'
misc.										poly
Total aberr (+ gaps)	1	1		0	0		2	0		2	0		34	27
Total aberr (- gaps)	1	1		0	0		2	0		2	0		34	25

^a)Abbreviations used for various types if aberrations are listed in APPENDIX 2

misc = (miscellaneous) aberrations not belonging to the ones mentioned above

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

*) Significantly different from control group (Fisher’s exact test), *P<0.05, **P<0.01 or ***P<0.001

 

Chromosome aberrations in human lymphocyte cultures treated with Formaldehyde, oligomeric reaction products with

acetone and diphenylamine in the presence of S9-mix in the first cytogenetic assay (3 h exposure time, 24 h fixation time)

Conc	DMSO (1.0% v/v)			17μg/ml			52μg/ml			164μg/ml			CP 10μg/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			88			83			78			44
No. of Cells scored	150	150	300	150	150	300	150	150	300	150	150	300	150	150	300
No. of Cells with aberrations (+ gaps)^a)	0	2	2	1	0	1	0	0	0	0	0	0	25	44	^***)69
No. of Cells with aberrations (- gaps)	0	1	1	1	0	1	0	0	0	0	0	0	25	43	^***)68
g'		1											1	2
g'’
b'		1		1									24	34
b'’													1	5
m'
m'’
exch.													1	7
dic.
d'
misc.					endo						5poly
Total aberr (+ gaps)	0	2		1	0		0	0		0	0		27	48
Total aberr (- gaps)	0	1		1	0		0	0		0	0		26	46

^a)Abbreviations used for various types if aberrations are listed in APPENDIX 2

misc = (miscellaneous) aberrations not belonging to the ones mentioned above

The numerical variations endoreduplications (endo) and polyploidy (poly) were not counted as an aberration

*) Significantly different from control group (Fisher’s exact test), *P<0.05, **P<0.01 or ***P<0.001

 

Mitotic index of human lymphocyte cultures treated with Formaldehyde, oligomeric

reaction products with acetone and diphenylamine in the second cytogenetic assay

Test item concentration (μg/ml)	Number of metaphases^a)
	Absolute	Number of cells scored	Percentage of control
Without metabolic activation (-S9-mix)
24 h exposure time, 24 h fixation time
Control^b) 17 52 164^c) MMC-C; 0.2μg/ml MMC-C; 0.3μg/ml	86 – 70 74 – 67 61 – 58 45 – 42 29 – 32 33 – 35	1011 – 1034 1006 – 1027 1010 – 1029 1005 – 1013 1029 – 1005 1022 – 1025	100 90 76 56 39 44
48 h exposure time, 48 h fixation time
Control^b) 17 52 164^c) MMC-C; 0.1μg/ml MMC-C; 0.15μg/ml	75 – 64 59 – 60 51 – 46 34 – 38 33 – 36 40 – 31	1027 – 1014 1019 – 1003 1039 – 1013 1022 – 1018 1002 – 1004 1020 – 1016	100 86 70 52 50 51

a) Duplicate cultures

b) Ethanol

c) Formaldehyde, oligomeric reaction products with acetone and diphenylamine precipitated in the culture medium

 

Chromosome aberrations in human lymphocyte cultures treated with Formaldehyde, oligomeric reaction products with

acetone and diphenylamine in the absence of S9-mix in the second cytogenetic assay (24 h exposure time, 24 h fixation time)

Conc	DMSO (1.0% v/v)			17μg/ml			52μg/ml			164μg/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			90			76			56			39
No. of Cells scored	150	150	300	150	150	300	150	150	300	150	150	300	75	150	225
No. of Cells with aberrations (+ gaps)^a)	2	2	4	1	2	3	1	2	3	1	0	1	38	46	^***)84
No. of Cells with aberrations (- gaps)	2	2	4	1	2	3	1	0	1	1	0	1	38	46	^***)84
g'
g'’								2
b'	2	2		1						1			26	31
b'’					2		1						17	13
m'
m'’
exch.													3	5
dic.
d'
misc.								poly
Total aberr (+ gaps)	2	2		1	2		1	2		1	0		46	49
Total aberr (- gaps)	2	2		1	2		1	0		1	0		46	49

^a)Abbreviations used for various types if aberrations are listed in APPENDIX 2

misc = (miscellaneous) aberrations not belonging to the ones mentioned above

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

*) Significantly different from control group (Fisher’s exact test), *P<0.05, **P<0.01 or ***P<0.001

 

Chromosome aberrations in human lymphocyte cultures treated with Formaldehyde, oligomeric reaction products with

acetone and diphenylamine in the absence if S9-mix in the first cytogenetic assay (48 h exposure time, 48 h fixation time)

Conc	DMSO (1.0% v/v)			17μg/ml			52μg/ml			164μg/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			86			70			52			50
No. of Cells scored	150	150	300	150	150	300	150	150	300	150	150	300	75	75	150
No. of Cells with aberrations (+ gaps)^a)	1	1	2	3	0	3	1	3	4	4	2	6	49	40	^***)89
No. of Cells with aberrations (- gaps)	1	1	2	3	0	3	1	3	4	4	2	6	49	40	^***)89
g'
g'’
b'	1	1		2			1	3		3	1		45	32
b'’				1						1	1		11	7
m'
m'’													1	3
exch.													11	11
dic.
d'
misc.							poly	poly
Total aberr (+ gaps)	1	1		3	0		1	3		4	2		68	53
Total aberr (- gaps)	1	1		3	0		1	3		4	2		68	53

^a)Abbreviations used for various types if aberrations are listed in APPENDIX 2

misc = (miscellaneous) aberrations not belonging to the ones mentioned above

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

*) Significantly different from control group (Fisher’s exact test), *P<0.05, **P<0.01 or ***P<0.001

Applicant's summary and conclusion

Conclusions:

Formaldehyde, oligomeric reaction products with acetone and diphenylamine is not clastogenic in human lymphocytes under the experimental conditions described in the report.

Executive summary:

Evaluation of the ability of Formaldehyde, oligomeric reaction products with acetone and diphenylamine to induce chromosome aberrations in cultured peripheral human lymphocytes.

 

The report describes the effect of Formaldehyde, oligomeric reaction products with acetone and diphenylamine 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 Formaldehyde, oligomeric reaction products with acetone and diphenylamine was tested in two independent experiments.

 

The study procedures described in the report are in compliance with the following guideline:

Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Guideline no. 473: In Vitro Mammalian Chromosome Aberration Test (adopted September 26, 2014).

 

Batch IC5B04P006 of Formaldehyde, oligomeric reaction products with acetone and diphenylamine consisted of dark brown flakes. The test item was dissolved in dimethyl sulfoxide.

 

In the first cytogenetic assay, the test item was tested up to 164 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 item precipitated in the culture medium at this dose level.

 

In the second cytogenetic assay, the test item was tested again up to 164 μg/ml for a 24 and 48 h continuous exposure time with a 24 and 48 h fixation time in the absence of S9-mix. The test item 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 95% control limits of the distribution of the historical negative control database. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. 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.

 

Formaldehyde, oligomeric reaction products with acetone and diphenylamine 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 Formaldehyde, oligomeric reaction products with acetone and diphenylamine 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 Formaldehyde, oligomeric reaction products with acetone and diphenylamine 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 this test is valid and that Formaldehyde, oligomeric reaction products with acetone and diphenylamine is not clastogenic in human lymphocytes under the experimental conditions described in the report.